THE  LIBRARY 

OF 

THE  UNIVERSITY 
OF  CALIFORNIA 

LOS  ANGELES 


GIFT 

Mrs.  Lawrence  Stuppy 


BLOOD  AND  URINE  CHEMISTRY 


A 

m 


2  _  3 

PI.ATF,    I. — STANDARD    WEDGES. 

1.  Standard  Phenolsulphonphthalein    Wedge. 

2.  Standard  Uric   Acid   Wedge. 

3.  Standard  Nitrogen  Wedge. 

4.  Standard  Cholesterol    Wedge. 


THE  NEWER  METHODS 
OF 

BLOOD  AND  URINE 
CHEMISTRY 


BY 


E.  B.  H.  GRADWOHL,  M.D. 

DIRECTOR  OF  THE  PASTEUR  INSTITUTE  OF  ST.  LOUIS  AND  THE  GRADWOHL 
BIOLOGICAL    LABORATORIES,    ST.    LOUIS 


AND 


A.  J.  BLAIVAS 

ASSISTANT    IN    THE    SAME;    SOMETIME    TECHNICIAN    IN    PATHOLOGICAL    CHEMICAL 

LABORATORIES,    NEW    YORK    POST-GRADUATE    MEDICAL    SCHOOL    AND 

HOSPITAL ;  AND  FORMER  ASSISTANT,  CHEMICAL  LABORATORY, 

ST.    LUKE'S    HOSPITAL,   NEW   YORK    CITY. 


WITH  SIXTY-FIVE  ILLUSTRATIONS  AND 
FOUR  COLOR  PLATES 


ST.  LOUIS 
C.  V.  MOSBY  COMPANY 

1917 


COPYRIGHT,  1917,  BY  THE  C.  V.  MOSBY  COMPANY 


Press  of 

The  C.  V.  Mosby  Company 
St.  Louis 


TO 
WILLIAM  MARION  REEDY 

AN  ESTEEMED  FRIEND 


CxY 
5 

7 
11/7 


PREFACE 

The  present  work  was  undertaken  in  response  to  a  demand  from 
our  many  professional  friends  who  have  become  keenly  inter- 
ested in  this  line  of  laboratory  investigation.  We  lay  but  little 
claim  to  originality  but  feel  that  if  we  have  collected  the  major 
part  of  the  information  that  is  so  widely  scattered  throughout 
the  journal  literature  of  the  past  three  or  four  years,  and  boiled 
it  down  into  a  readily  digested  form,  our  labors  will  not  have 
been  in  vain.  The  investigations  in  blood  chemistry  are  pro- 
ceeding rapidly  so  that,  of  necessity,  this  sort  of  book  will  be 
difficult  to  keep  up-to-date.  We,  therefore,  ask  for  the  indul- 
gence of  those  who  are  insistent  upon  the  very  last  word. 

It  will  be  noted  that,  in  the  main,  we  have  given  but  one 
method  for  each  test.  We  have  done  this,  because  we  believe  we 
know  what  the  majority  of  the  practical  workers  along  this  line 
judge  the  best  test  to  be :  besides,  we  see  no  reason  for  describing 
tests  that  time  and  experience  have  proved  fallacious  or  too  com- 
plicated. The  work  in  hand  gives  the  tcchnic  just  as  we  carry 
out  our  routine  and  research  work  in  our  laboratories. 

K,  B.  H.  G. 
A.   J.   B. 

St.  Louis,  Mo. 


CONTENTS 


PART  I. 
TECHNIC  OF  BLOOD  CHEMISTRY. 

CHAPTER  I.                                                 PAGE 
GENERAL  CONSIDERATIONS 17 

CHAPTER  II. 
SUGAR  IN   BLOOD 28 

CHAPTER  III. 
CREATININE 34 

CHAPTER  IV. 
CREATINE 36 

CHAPTER  V. 
URIC  ACID 37 

CHAPTER  VI. 
UREA   . 42 

CHAPTER  VII. 

NONPROTEIN  NITROGEN 47 

/ 

CHAPTER  VIII. 
CHOLESTEROL 50 

CHAPTER  IX. 
TOTAL  SOLIDS 53 

CHAPTER  X. 
TOTAL  NITROGEN i,    .    .    54 

CHAPTER  XI. 
CHLORIDES 57 

CHAPTER  XII. 

VAN  SLYKE  METHOD  FOR  THE  DETERMINATION  OF  THE  CARBON  DIOXIDE 

COMBINING  POWER  OF  BLOOD  PLASMA 59 


12  CONTENTS 

PART  II. 
CHEMICAL  ANALYSIS  OF  URINE. 

CHAPTER  XIII. 
TOTAL,  NITROGEN 76 

CHAPTER  XIV. 
UREA 79 

CHAPTER  XV. 
AMMONIA  82 

CHAPTER  XVI. 
URIC  ACID 84 

CHAPTER  XVII. 
CREATININE 86 

CHAPTER  XVIII. 

CREATINE  .     .     .     .     .     .     .     .     . .     .     .     .     .     .     .     88 


CHAPTER  XIX. 
PHENOLSULPHONPHTHALEIN  89 


CHAPTER  XX. 
CHLORIDES 95 


CHAPTER  XXI. 
GENERAL  ANALYSIS     ,  96 


CHAPTER  XXII. 

MICROSCOPIC  ANALYSIS  OF  URINARY  SEDIMENTS     .  .     109 


CHAPTER  XXIII. 
THE  STAINING  OF  BACTERIA  IN  URINE     .     .    . 


CHAPTER  XXIV. 

DESCRIPTION  OF  THE  COLORIMETER    .  .     132 


CONTEXTS  13 

PART  III. 
BLOOD  FINDINGS  AND  THEIR  INTERPRETATION. 

CHAPTER  XXV. 
BLOOD   SUGAR ' 139 

CHAPTER  XXVI. 
AciDOSlS : 165 

CHAPTER  XXVII. 
BLOOD  CHANGES  IN  GOUT 194 

CHAPTER  XXVIII. 

BLOOD  CHEMISTRY  AND  NEPHRITIS .    201 


ILLUSTRATIONS 

PLATE      I. — STANDARD  WEDGES Frontispiece 

PLATE    II. — STANDARD  WEDGES Facing  page  28 

PLATE  III. — URINE    COLOR    REACTIONS Facing  page  96 

PLATE  IV. — BENEDICT'S  TEST  FOR  SUGAR Facing  page  100 

FIG.  PAGE 

1.  View  of  one  side  of  chemical  laboratory  showing  balance,  dessicator, 

etc 20 

2.  View  of  another  side  of  chemical  laboratory  showing  Van  Slyke's 

carbon  dioxide  apparatus  and  the  urea  apparatus  set  up  and 

connected  to  the  suction 21 

3.  Blood  chemical  table  showing  urea  apparatus  and  water-bath  used 

for  the  uric  acid  determinations 22 

4.  Showing  a  high  power  centrifuge  placed  so  as  to  economize  space     .  25 

5.  Manner  of  procuring  blood 26 

6.  Gradwohl  tourniquet 27 

7.  Chemical  blood  bottle 27 

8.  50   c.c.   centrifuge   tube 28 

9.  Ostwald  pipette 29 

10.  Graduated  sugar  tube 29 

11.  Showing  sugar  tube  immersed  in  a  beaker  of  water 30 

12.  Casserole 37 

13.  Showing  centrifuge  tube  attached  to  suction 38 

14.  Volumetric  flask 39 

15.  Showing  the  urea  apparatus  set  up  and  connected  to  suction     ...  43 

16.  Microburner .  47 

17.  Apparatus  for  removing  fumes  in   connection  with  nitrogen   deter- 

minations     48 

18.  Weighing  bottle  for  total  solids 53 

19.  Kjeldahl  flask 54 

20.  Digestion   rack       . 55 

21.  Kjeldahl  apparatus  showing  condenser 55 

22.  Graduated  centrifuge  tube 57 

23.  Showing  operator  saturating  blood  plasma  with  carbon  dioxide     .     .  60 

24.  CO,  apparatus 61 

25.  Dropping  bottles  for  use  in  connection  with  CO,  determination     .     .  62 

26.  CO2  apparatus  showing  air  being  forced  out 64 

27.  CO,  apparatus.     Mercury  should  not  go  below  mark  X 65 

28.  Phenolsulphonphthalein  ampule 90 

29.  Graduated  syringe  used  for  the  injection  of  phenolsulphonphthalein  90 

30.  Urinometer 99 

31.  Showing  Benedict's  method  for  the  quantitative  estimation  of  sugar  101 

32.  Graduated   conical   centrifuge    tube 103 

33.  Porcelain  tablet  for  the  determination  of  phosphates 107 

34A.  Centrifuge        109 

34B.  Conical  centrifuge  tube 109 

35A.  Granular   casts .  Ill 


16  ILLUSTRATIONS 

FIG.  PAGE 

35B.  Granular  casts Ill 

36.  Hyaline   casts 112 

37A.  Epithelial  casts 112 

37B.  Epithelial  casts 112 

38.  (a)  Blood  casts  (yellow  in  color)  ;   (b)  Pus  casts 113 

39.  Fatty  casts    . 113 

40A.  Cyiindroids 114 

40B.  Cyiindroids 114 

41.  Erythrocytes 115 

42.  Human    spermatozoa 115 

43.  "Triple    Phosphate"      . 117 

44.  Calcium  oxalate  crystals 118 

45.  Calcium  phosphate  crystals 119 

46.  Calcium   sulphate   . 120 

47.  Calcium   carbonate   crystals 120 

48.  Uric    acid    crystals 121 

S49.  Acid  sodium  urate  crystals 122 

50.  Ammonium   urate   crystals 122 

51.  Cholesterol    crystals 123 

52.  Hippuric  acid  crystals 124 

53.  Crystals  of   impure  leucine 125 

54.  Representation  of  Hellige   colorimeter 133 

55.  Representation   of  Hellige   colorimeter 134 

56.  Representation   of   Hellige   colorimeter 135 

57.  Representation  of  Hellige  colorimeter 136 

58.  Optical  arrangement  of  window  of  colorimeter 137 

59.  Diagram  illustrating  normal  sugar  metabolism 143 

60.  Diagram  illustrating  the  nonutilization  of  sugar  in  diabetes     .     .     .  143 

61.  Diagram  illustrating  excessive  formation  of  sugar  through  nonreten- 

tion  of  glycogen  in  the  liver 144 

62.  Fridericia  apparatus  for  determination  of  carbon  dioxide  in  alveolar 

air .     .  173 

63.  The  characteristic  blood  pictures  in  gout,  diabetes,  and  nephritis     .  202 

64.  The  characteristic  blood  pictures  in  gout,  diabetes,  and  nephritis     .  203 

65.  Blood  and  urine  findings  in  thermic  fever 213 


BLOOD  AND  URINE  CHEMISTRY 


PART  I. 
TECHNIC  OF  BLOOD  CHEMISTRY 


CHAPTER  I. 
GENERAL  CONSIDERATIONS. 

Chemical  analyses  of  blood  have  for  years  been  looked  upon 
as  belonging  to  experimental  physiological  chemistry,  and,  in  no 
sense  of  practical  use  such  as  are  urinary  analyses,  gastric  con- 
tents analyses,  etc.  As  bedside  ai,ds  to  diagnosis,  blood  chemical 
analyses  did  not  really  exist  until  the  epoch-making  work  of 
Folin  brought  the  question  to  the  very  forefront  of  medical  litera- 
ture. It  was  Folin  who  called  attention  to  the  practicability  of 
making  blood  chemical  tests  with  the  idea  in  view  of  aiding  the 
physician  in  diagnosis,  using  ' '  microchemical ' '  methods  which  have 
proved  successful  in  quantitative  analytical  chemistry.  His  work 
has  been  followed  by  others  who  have  simplified  some  of  the  meth- 
ods. Such  eminent  authorities  as  Folin  and  Denis,  Benedict  and 
Lewis,  and  Myers  and  Fine  deserve  much  credit  for  introducing 
these  new  and  reliable  methods  of  clinical  laboratory  technic. 

It  might  be  asked  here,  of  what  practical  use  is  blood  chemistry ; 
what  additional  information  can  it  give  us  over  the  tried  and  ac- 
cepted methods  of  urinary  analyses?  Are  the  data  obtainable 
from  blood  chemical  manipulations  of  more  service  to  the  diag- 
nostician than  are  urinary  findings?  Does  blood  chemistry  give 
data  not  hitherto  obtainable  with  urine  chemical  methods?  "We 
must  emphatically  answer  "yes,"  to  both  questions.  In  fact,  we 
trust  that  the  reader  will  recognize,  after  the  perusal  of  this 
book,  that  blood  chemical  analyses  far  surpass  in  value  the  most 


18  BLOOD   AND    URINE    CHEMISTRY 

exact  and  intricate  qualitative  and  quantitative  urinary  analyses. 
We  aim  to  convince  the  reader  that  of  the  two  sets  of  facts,  one 
furnished  by  urine  analyses,  the  other,  by  blood  analyses,  the  lat- 
ter is  of  far  greater  importance.  We  do  not  wish  to  decry,  for 
a  moment,  the  carrying  out  of  routine  urinary  analyses,  nor  do 
we  wish  to  minimize  the  splendid  helpfulness  of  a  good  urine 
analysis:  rather,  do  we  say  that  blood  and  urine  investigations 
should  go  hand  in  hand,  but  that  the  information  obtainable 
from  the  blood  chemical  analysis,  being  of  a  different  character, 
representing  estimation  of  retained  products  of  metabolism 
rather  than  the  estimation  of  pathologically  changed  ingredients 
of  a  fluid  such  as  a  search  for  albumin  or  sugar  in  urine  implies, 
gives  a  far  better  idea  of  metabolic  changes  and  furnishes  a  superior 
basis  for  the  diagnostic  and  prognostic  evaluation  of  a  case  to  that 
furnished  by  the  urine  analyses.  The  blood  chemical  analysis 
tells  us  what  the  blood  is  storing  up,  what  the  kidneys  are  doing  and 
what  they  are  not  doing,  and  also  the  exact  status  of  nitrogenous 
and  carbohydrate  equilibrium.  The  urine  analysis  tells  us  a 
great  deal  about  the  pathology  of  the  kidney  function.  One 
might  be  described  as  an  estimation  of  the  organic  changes  in 
the  kidneys;  the  other,  the  blood  chemical  analysis,  is  an  estima- 
tion of  the  minuticB  of  the  renal  function,  from  a  pathological 
chemical  and  a  pathological  physiological  viewpoint.  Undue  ex- 
cretion of  sugar  in  the  urine  is  pathological,  but  how  about  the 
interpretation  of  the  finding  of  glycosuria?  We  know  that  the 
amount  of  sugar  in  the  blood  gives  a  far  better  picture  of  carbo- 
hydrate metabolism  than  does  the  appearance  of  sugar  in  the  urine. 
Sugar  appears  in  the  urine  in  a  case  of  diabetes  mellitus  purely  as 
an  "overflow"  proposition,  whereas  there  may  be  an  enormous 
sugar  retention  in  the  blood  beforfijfoe  kidneys  permit  it  to  leak 
jhrough.  Thus  an  individual  may  have  a  hyperglycemia  long 
before  he  has  a  glycosuria.  There  may  be  a  so-called  prediabetic 
stage  to  which  the  older  writers  often  referred;  only  a  blood 
chemical  estimation  of  sugar  would  detect  this.  Again,  there 
may  be  a  case  of  low  hyperglycemia  and  pronounced  glycosuria 
with  kidneys  in  individual  cases  readily  permeable  to  sugar. 
Glycosuria  in  this  case  would  give  one  no  idea  of  the  low  grade 
of  hyperglycemia.  In  renal  diabetes,  too,  there  is  no  hypergly- 


GENERAL    CONSIDERATIONS  19 

cemia,  simply  a  glycosuria  possibly  due  to  unusual  permeability  of 
the  kidneys  for  the  normal  blood  sugar,  never  a  hyp ergly cemia. 
How  could  one  differentiate  then  between  diabetes  mellitus  and 
renal  diabetes  without  a  cmnparative  blood  and  urine  chemical 
"allalysis1/  " 

We  feel  that  the  subject  has  now  been  sufficiently  worked  out 
to  demand  a  condensation  of  all  the  facts  gleaned  by  blood  chem- 
istry and  their  interpretation  in  clinical  medicine  into  a  small 
textbook  for  the  information  of  those  who  are  interested.  The 
literature  has  appeared  practically  in  only  the  technical  journals, 
principally  the  Journal  of  Biological  Chemistry.  These  articles 
are,  as  a  rule,  inaccessible  to  many  physicians  and  even  to  some 
of  the  laboratory  workers  in  communities  where  there  is  no  medi- 
cal library.  The  writers'  task  is,  therefore,  to  give  fully  the 
best  methods  that  have  been  devised  by  the  workers  in  this  field 
together  with  such  facts  as  they  themselves  have  gleaned  during 
years  of  effort,  together  with  the  most  important  literature  on 
this  question.  The  subject  is  under  close  investigation  and  rapid 
strides  are  being  made.  It,  therefore,  behooves  those  who  are 
interested  in  the  practical  and  scientific  sides  of  medicine  to  keep 
informed  on  all  this  progress.  We  trust  that  our  modest  efforts 
will  assist  in  spreading  the  facts  before  those  not  familiar  with 
them  and  that  others  may  be  stimulated  to  assist  in  this  work  of 
accurately  estimating  bodily  metabolism  in  health  and  in  disease. 

We  shall,  later  on  in  the  work,  give  our  interpretation  of  the 
technical  findings  in  blood  and  urine  chemistry.  Owing  to  the 
wide  interest  in  this  newborn  side  of  laboratory  diagnosis,  we 
wish  to  immediately  take  up  the  question  of  installation  of  the 
laboratory  for  this  sort  of  work  and  the  actual  technic  of  the  tests. 

Installation  of  the  Blood  and  Urine  Chemical  Laboratory. 

We  have  described  in  the  following  pages  the  various  apparatus, 
reagents,  glassware,  etc.,  needed  in  this  work.  We  shall,  as  it  were, 
construct  a  model  laboratory  for  the  reader  in  which  he  may 
most  profitably  pursue  these  investigations.  We  shall  not  enu- 
merate unnecessary  apparatus,  but  shall  endeavor  to  make  the 
wants  of  the  prospective  worker  as  few  as  possible.  Stately  halls, 
marble  columns,  and  lavish  expenditure  do  not  alone  imply  great 
work.  Simplicity,  modesty,  coupled  with  untiring  zeal  and  exact 


20 


BLOOD   AND   URINE    CHEMISTRY 


observation,  have  given  us  what  great  advances  medicine  today 
has  gained,  and  to  that  end  we  will  construct  a  practical  and  in- 
expensive laboratory  for  those  who  contemplate  launching  into 
this  department  of  laboratory  medicine. 

We  will  give  the  essentials  of  equipment  and  the  ideal  of  their 
arrangement,  allowing  the  ingenuity  and  particular  facilities  of 
each  worker  contemplating  taking  up  this  technic  to  work  out 
his  own  arrangement  of  laboratory  furniture,  etc. 

Selection  of  the  Room. — Preferably  a  room  should  be  selected 


Fig.  1. — View  of  one  side  of  chemical  laboratory  showing  balance,  dessicator,  etc. 

with  good  northern  exposure  for  the  accurate  reading  of  the 
colorimeter.  There  should  be  a  well  protected  place  for  the 
chemical  balance,  safe  from  sunlight  and  jarring.  There  should 
be,  also,  a  firm  block  of  wood  arranged  conveniently  for  the  plac- 
ing of  the  centrifuge.  There  should  be  running  water  in  the 
room  for  two  purposes;  one  for  suction  in  running  a  Chapman 
pump,  the  other  for  obtaining  water  for  a  water-bath,  cleaning 
glassware,  etc.  There  should  also  be  a  chemical  hood  with  the 
customary  outlet  for  permitting  vapors  to  escape.  There  should 


GENERAL    CONSIDERATIONS 


21 


also  be  a  convenient,  strongly  constructed  table  for  the  microscope 
and  balance.  This  in  a  general  way  covers  the  arrangement  of 
the  room  for  the  larger  articles.  In  addition  to  these  features, 
there  should  be  shelving  for  the  accommodation  of  the  reagent 
bottles,  with  drawers  and  cupboards  for  the  storage  of  glass- 
ware, tubing,  etc.  The  work  table  should  be  large  enough  to 
permit  from  two  to  six  Bunsen  burners  to  be  placed  in  rows  for 
the  simultaneous  heating  of  blood  specimens. 


Fig.  2. — View  of  another  side  of  chemical  laboratory  showing  Van  Slyke's  carbon  dioxide 
apparatus  and  the  urea  apparatus  set  up  and  connected  to  the  suction. 

For  the  purpose  of  illustrating  several  views  of  a  model  labora- 
tory, we  call  attention  to  Figs.  1  and  2  which  show  the  C02  ap- 
paratus, chemical  balance  set  up,  desiccator,  etc.  In  Fig.  3  is 
shown  the  blood  chemical  table  proper,  with  running  water  in 
the  middle  of  same,  the  Chapman  suction  pump,  and  the  water- 
bath  set  up  for  uric  acid  estimations.  It  also  shows  the  arrange- 
ment of  the  cylinders  for  urea  estimation,  a  complete  description 
of  which  wTill  be  found  in  the  chapter  on  this  subject  (see  p.  42). 


22 


BLOOD   AND   URINE    CHEMISTRY 


Fig.   3. — Blood  chemica 


showing  urea  apparatus 
acid  determinations. 


md   water-bath    used    for   the   uric 


Chemicals  and  Apparatus  Used  in  the  Newer  Chemical  Analysis 
of  Blood  and  Urine. 

It  is  essential  to  have  a  "Hellige"  colorimeter  which  is  de- 
scribed on  page  133  and  a  balance  which  is  accurate  to  one-tenth 
of  a  milligram. 


CHEMICALS. 
Urea  N. 

Urease,   10   gins. 
Mercuric   iodide,    200    gms. 
Potassium  iodide,   100   gms. 
Potassium   hydroxide,    400   gms. 
Amyl   alcohol,   100   c.c. 
Caprylic    alcohol,    25    c.c. 
Hydrochloric  acid,  500  gms. 


APPARATUS. 
Urea  N. 

6  Volumetric  flasks    (1000  c.c.,  500 

c.c.,  250  c.c.),  2  of  each. 
50  Test    tubes    about   200   mm.    long 
and  of   diameter  such   that  will 
slip  into  100  c.c.  graduate. 

2  Nests  of  beakers  from  50  c.c.  to 
1000  c.c.  capacity. 

1  Sulphuric  acid  wash  bottle. 

6  Bunsen  burners. 

6  Tripods. 

6  Pieces  wire  gauze,  asbestos  center. 

1  Thermometer. 


GENERAL    CONSIDERATIONS 


23 


CHEMICALS. 
Urea  N — Cont'd 


APPARATUS. 
Urea  N — Cont  'd 

3  Graduates,  100  c.e.  (no  lips),  non- 
graduated. 

3  Graduates,  100  c.c.  (no  lips),  grad- 

uated. 

4  Volumetric  flasks   (50  c.c.). 

9  Pipettes,  5  c.c.,  20  c.c.,  25  c.c.  (3 

of  each). 
6  Two-hole    rubber    stoppers    to    fit 

graduates. 
1  Twenty-four    foot    tubing    to    fit 

holes. 

1  Suction  pump. 
1  Desiccator. 
1  Wash  bottle  and  connection. 


Uric  acid. 

Acetic    acid,    500    gms. 
Alumina  cream,  250  gms. 
Potassium   cyanide,   30    gms. 
Silver  nitrate,  30  gms. 
Magnesium   sulphate,    50    gms. 
Ammonium  chloride,  100  gms. 
Ammonia    (cone.),   500   gms. 
Uric   acid    (Kahlbaum),    i/2   gm. 
Sodium  tungstate,   100   gms. 
Hydrogen  disodium  phosphate, 

25  gms. 
Dihydrogen   sodium    phosphate, 

5  gms. 
Sodium  carbonate,  500  gms. 


Uric  acid. 

4  Cylinders,     (100    c.c.).       (Gradu- 
ated.) 

6  Casseroles,  (375  c.c.  capacity). 
12  Stirring  rods,  (6  in.). 

1  Water-bath. 

6  Funnels,  about  4  in.  diameter. 
100  Filter    papers    (for    above    fun- 
nels) . 

6  Centrifuge  tubes,  (15  e.c.,  conical). 

1  Centrifuge. 

8  Pipettes,  2  c.c.  and  10  c.c.— four 
of  each. 

1  Wash  bottle  and  connection    (for 
hot  water). 


Sugar. 

Picramic  acid,  %  gm. 
Picric  acid,  100  gms. 

Creatine  and  Creatinvne. 
Potassium  bichromate,  25  gms. 
Creatinine,   %   gm. 
Sodium    hydroxide,    500    gms. 


Sugar. 

6  Sugar  tubes,  graduated  to  20  c.c. 
3  Pipettes,  1  c.c.  and  3  c.c. 

Creatine  and  Creatinine. 
8  Graduates,   10   c.c.,   25   c.c. — 4   of 

each. 

3  Pipettes,  1  c.c.  graduated  1/100. 
12  Centrifuge  tubes,  15  c.c.  and  50 

c.c. — six  of  each. 
1  Autoclave. 


C02  Combining  Power  of  Plasma. 
Phenolphthalein,  10  gms. 
Sulphuric  acid,  500  gms. 
Mercury,   5   Ibs. 
Caprylic  alcohol,   30  c.c. 


C02  Combining  Power  of  Plasma. 
1  Van  Slyke  apparatus. 
1  Heavy  stand  and  rod. 
1  6-ft.  Heavy  suction  tubing. 
1  Iron  rod  and  connection. 


24 


BLOOD   AND   URINE    CHEMISTRY 


CHEMICALS. 

C02    Combining   Power   of  Plasma — 
Cont'd. 


Nonprotein   Nitrogen. 
Potassium  sulphate,  50  gms. 
Copper  sulphate,   50   gms. 
Trichloracetic  acid,  100  c.c. 
Kaolin,  25  gms. 

Cholesterol. 
Cnloroform,    500    c.e. 
Acetic  anhydride,  50  c.c. 
Cholesterol   or  naphthol, 
Green  B,  1  gm. 
Ether,  250   c.c. 
Alcohol   (redistilled),  500  c.c. 

Total  Solids. 


Chlorides. 

Colloidal   iron,   50    c.c. 
Potassium  chromate,  25  gms. 
Silver  nitrate,  10  gms. 
Ferric  ammonium  sulphate,  100  gms. 
Nitric  acid,  500  gms. 
Ammonium  thyocyanate,  10  gms. 

Total  Nitrogen. 
Congo  red,  5  gms. 
Peroxide  of  hydrogen,  50  c.c. 


Phenol  phtlialein. 
Phenolsulphonphthalein  in  1  c.c. 
ampules — 3. 

Ammonia. 
Included  in  foregoing. 


APPARATUS. 

C02   Combining   Power   of   Plasma — 
Cent  'd. 

1  Large  clamp  and  connection. 

2  Eings. 

6  Dropping  bottles  (with  rubber  nip- 
pies). 

1  Separating  funnel. 

1  Apparatus  for  saturating  blood 
plasma  (consisting  of  bottle  filled 
with  glass  beads  and  connection). 

Nonprotein  Nitrogen. 

3  Microburners. 

1  Apparatus    for    removing    fumes 

(large  bottle,  2-hole  rubber  stop- 
per and  connection,  1  stand  and 
connection) . 

Cholesterol. 

3  100  c.c.  graduated  flasks. 
3  25  c.c.  beakers. 

2  10  c.c.  glass-stoppered,  graduated 

cylinders. 


Total  Solids. 

2  Weighing  bottles    (glass  stoppers 

and  block  of   filter   and  connec- 
tion). 

Chlorides. 

3  Evaporating    dishes,    50    c.c.    ca- 

pacity. 

2  Volumetric  flasks,  25  c.c.  capacity. 
2  Burettes,  stand  and  connection. 


Total  Nitrogen. 

2  Kjeldahl  flasks. 

1  Digestion  rack,  consisting  of  out- 
let for  fumes,  distilling  outfit, 
and  receiving  bottle. 


Phenolphthalein. 
2  Graduates,  1000  c.c. 
1  Accurately  graduated  1  c.c. 
syringe  with  needles. 

Ammonia. 
Included  in  foregoing. 


GENERAL    CONSIDERATIONS 


25 


A  high  power  centrifuge  is  advisable,  one  that  can  carry  15 
c,c.,  50  c.c.,  and  100  c.c.  centrifuge  tubes.  Fig.  4  illustrates  a  con- 
venient method  of  placing  the  centrifuge  so  as  to  economize  space. 
The  centrifuge  is  set  on  heavy  blocks  of  wood  so  as  to  avoid  un- 
due vibration.  The  work  table  is  hinged  so  as  to  utilize  the 
space  occupied  by  the  centrifuge. 


Fig.   4.- — Showing  a  high  power  centrifuge  placed  so  as  to   economize   space. 

Manner  of  Procuring  and  Handling  of  Blood. 

The  withdrawal  of  blood  can  best  be  accomplished  by  following 
the  method  of  one  of  the  writers  (Gradwohl)  in  obtaining  blood 
for  the  Wassermann  reaction  (see  Fig.  5),  which  is  as  follows: 

Expose  the  bend  of  the  elbow  where  a  prominent  vein  can  usu- 
ally be  found.  In  women  and  men  with  a  good  deal  of  adipose 
tissue,  these  veins  are  sometimes  not  visible.  In  such  cases,  select 
the  wrist  or  back  of  the  hand.  Place  a  tourniquet,  either  bandage 
or  rubber  tubing,  above  the  bend  of  the  elbow.  The  patient  is 
then  instructed  to  double  his  fist,  which  still  further  assists  in 


26  BLOOD   AND   URINE    CHEMISTRY    ' 

distending  the  veins  between  the  fist  and  the  portion  of  the  arm 
upon  which  the  tourniquet  is  tied. 

The  skin  over  the  vein  is  then  thoroughly  cleansed  by  rubbing 
vigorously  with  alcohol.  Although  iodine  is  a  good  antiseptic, 
it  is  not  advisable  to  use  it,  as  it  leaves  a  dark  stain  on  the  skin 
which  obscures  the  vein  and  makes  it  difficult  to  find. 

The  needle  is  then  removed  from  the  test  tube  and  plunged 
into  the  vein,  procuring  at  least  25  c.c.  of  blood  in  this  manner. 


Fig.  5. — Manner  of  procuring  blood. 

At  this  point  we  might  call  attention  to  the  usefulness  of  the 
Gradwohl  tourniquet  (Fig.  6)  in  blood  withdrawal.  This  gives 
uniform  compression  and  readily  permits  one  to  liberate  the 
tourniquet  without  dislodging  the  needle  from  the  vein.  By 
alternately  releasing  and  clamping  the  tourniquet,  sufficient  blood 
may  be  obtained  by  this  means  for  a  complete  chemical  analysis. 

The  blood  should  be  taken  in  the  morning,  before  breakfast.  In 
other  words,  if  it  is  not  convenient  to  take  the  blood  before  the 
usual  breakfast  hour,  then  it  may  be  taken  later,  but  the  patient 


GENERAL    CONSIDERATIONS 


27 


must  not  eat  anything  until  after  the  blood  is  taken.  The  reason 
for  this  is  that  all  data  on  the  normal  standards  and  the  patho- 
logical changes  have  been  obtained  with  blood  obtained  under 
these  conditions.  Therefore,  for  the  sake  of  uniformity,  we 
would  recommend  this  method. 

Amount  of  Blood  Needed. — Twenty-five  cubic  centimeters  of 
blood  should  be  withdrawn  for  a  complete  analysis. 


Fig.  6. — Gradwohl  tourniquet. 

This  blood  is  allowed  to  run  from  the  needle  into  small  chemi- 
cal bottles  (see  Fig.  7)  containing  10  drops  of  20  per  cent  solu- 
tion of  potassium  oxalate.  This  oxalate  should  be  previously 
dried  in  the  oven  over  night  at  100°  C. 


Fig.   7.— Chemical  blood  bottle. 

As  soon  as  possible  after  the  25  c.c.  of  blood  have  been  obtained, 
one  should  quickly  close  the  bottle  and  begin  shaking  vigorously 
so  as  to  complete  the  defibrination  of  the  blood  which  the  potas- 
sium oxalate  partially  accomplishes.  Do  not  stop  shaking  until 
perfect  fluidity  of  the  blood  has  been  obtained.  After  defibrina- 
tion of  the  blood,  the  process  of  chemical  analysis  should  begin. 


CHAPTER  II. 
SUGAR  IN  BLOOD. 

It  is  advisable  to  begin  the  blood  chemical  analysis  by  estima- 
tion of  sugar  and  creatinine  first,  because  these  two  substances 
most  quickly  deteriorate  and  hence  their  estimation  should  be 
begun  at  once.  Urea  and  uric  acid  determinations  can  be  done 
later. 

Take  a  50  c.c.  centrifuge  tube  (Fig.  8)  and  place  in  it  20  c.c. 
of  distilled  water.  Suck  up  5  c.c.  of  the  blood  into  an  Ostwald 


Fig.   8. — 50  c.c.   centrifuge  tube. 

pipette  (Fig.  9)  and  allow  it  to  run  into  the  bottom  of  the  centri- 
fuge tube,  below  the  water.  "Wash  the  pipette  by  alternating  draw- 
ing up  and  blowing  down  this  blood  and  water  mixture.  Star 
the  mixture  to  lake  the  cells.  Add  0.5  gram  dry  picric  acid 
which  precipitates  the  protein.  Stir  thoroughly.  Allow  it  to  stand 
10  to  15  minutes.  Stir  occasionally.  Place  in  centrifuge  and 
run  for  5  minutes  at  about  1500  revolutions  per  minute.  Now  re- 
move the  tube  from  the  centrifuge  and  filter  the  mixture  through 
a  small  filter  paper  into  a  clean,  dry  test  tube.  Part  of  this 
filtrate  is  used  for  the  sugar  estimation  and  part  for  the  creatin- 
ine estimation.  Take  3  c.c.  of  the  filtrate  for  the  sugar  test,  the 
remainder  being  reserved  for  the  creatinine  test.  Place  S^.c.  of 
the  filtrate  in  a  sugar  tube  (Fig.  10)  ;  add  1  c.c.  of  saturated  solu- 


PLATE    II. — STANDARD    WEDGES. 

1.  Standard    Picramic    Acid    Wedge. 

2.  Standard    Bichromate    (Normal.)    Wedge. 

3.  Standard   Creatinine   Wedge. 


SUGAR  IN   BLOOD 


29 


tion  of  sodium  carbonate,1  and  mix.  Immerse  the  test  tube  contain- 
ing this  mixture  in  a  large  beaker  of  water  and  then  boil  the  beaker 
over  a  free  flame  for  15  minutes  (Fig.  11),  after  which  it  is  al- 


cc 

-1C 


-(0 


Fig.   9. — Ostwald   pipette. 


Fig.   10. — Graduated  sugar  tube. 


lowed  to  cool.  The  final  step  in  the  test  is  to  so  dilute  this  cooled 
solution  with  distilled  water  that  it  will  be  weaker  in  color  than 
the  standard  picramic  acid  solution  with  which  it  is  to  be  com- 
pared in  the  colorimeter.  To  this  end  we  dilute  it  to  10,  15,  or 

1This  is  prepared  by  dissolving  220  grams  of  anhydrous  sodium  carbonate  in   1000  c.c. 
of  distilled  water. 


30 


BLOOD   AND   URINE    CHEMISTRY 


20  c.c.  [see  marks  upon  the  graduated  sugar  tube  (Fig.  10)].  It 
must  be  remembered  that  in  normal  cases  a  dilution  up  to  10  c.c.  will 
suffice,  but  beyond  this  it  is  often  necessary  to  dilute  to  15  c.c. 
or  even  20  c.c.  in  cases  of  hyperglycemia.  It  is  now  compared 


Fig.  11. — Showing  sugar  tube  immersed  in  a  beaker  of  water. 

in  the  colorimete^with  the  wedge  of  standard  picramic  acid. 
(See  Plate  II  for  the  color  of  the  standard  picramic  acid  wedge. )\ 

The  standard  picramic  acid  solution  is  a  staple  solution  and 
is  made  as  follows:  Dissolve  0.1  gm.  picramic  acid  and  0.2  gm. 
anhydrous  sodium  carbonate  in  30  c.c.  warm  distilled  water  and 
dilute  to  1  liter. 


SUGAR   IN   BLOOD  31 

Example  1. — Now  let  us  assume  that  the  reading  with  the  colori- 
meter was  52.  If  your  dilution  is  10,  subtract  52  from  100  which 
equals  48.  With  a  dilution  of  10,  multiply  this  by  0.002  which 
equals  0.096,  which  means  0.096%  of  sugar  present.  This  would  be 
a  normal  finding. 

Example  2. — Let  us  assume  that  the  reading  is  41  and  the 
dilution  is  25.  41  from  100  equals  59.  Multiply  this  by  0.005  which 
equals  0.295  (hyperglycemia).  In  other  words,  with  a  dilution 
of  10,  multiply  the  difference  between  the  reading  and  100  by 
0.002;  if  dilution  is  15,  multiply  the  difference  by  0.003;  if  the 
dilution  is  20,  multiply  by  0.004;  if  the  dilution  is  25,  multiply 
by  0.005;  etc. 

Identical  results  may  be  obtained  by  using  the  data  presented 
in  Table  I,  providing  the  estimation  was  made  on  the  basis  of  a 
dilution  of  10.  If  it  was  diluted  to  15  c.c.,  multiply  the  result  by 
1.5 ;  to  20  c.c.,  multiply  by  2 ;  etc. 

TABLE  I2 

ESTIMATION  OF  BLOOD  SUGAR  WITH  HELLIGE  COLORIMETER 


COLORI- 

BLOOD 

COLORI- 

BLOOD 

COLORI- 

BLOOD 

METRIC 

SUGAR  IN 

METRIC 

SUGAR  IN 

METRIC 

SUGAR  IN 

READING 

PER   CENT 

READING 

PER   CENT 

READING 

PER  CENT 

25 

0.150 

45 

0.110 

65 

0.070 

26 

0.148 

46 

0.108 

66 

0.068 

27 

0.146 

47 

0.106 

67 

0.066 

28 

0.144 

48 

0.104 

68 

0.064 

29 

0.142 

49 

0.102 

69 

0.062 

30 

0.140 

50 

0.100 

70 

0.060 

31 

0.138 

51 

0.098 

71 

0.058 

32 

0.136 

52 

0.096 

72 

0.056 

33 

0.134 

53 

0.094 

73 

0.054 

34 

0.132 

54 

0.092 

74 

0.052 

35 

0.130 

55 

0.090 

75 

0.050 

36 

0.128 

56 

0.088 

76 

0.048 

37 

0.126 

57 

0.086 

77 

0.046 

38 

0.124 

58 

0.084 

78 

0.044 

39 

0.122 

59 

0.082 

79 

0.042 

40 

0.020 

60 

0.080 

80 

0.040 

41 

0.118 

61 

0.078 

81 

0.038 

-   42 

0.116 

62 

0.076 

82 

0.036 

43 

0.114 

63 

0.074     . 

83 

0.034 

44 

0.112 

64 

0.072 

84 

0.032 

2Myers   and    Fine:      Chemical    Composition    of   the    Blood    in    Health   and    Disease,    New 
York,  1915. 


32  BLOOD   AND   URINE    CHEMISTRY 

The  authors  wish  to  caution  the  beginner  in  this  work  to  make 
his  readings  as  quickly  as  possible  as  these  colors  deteriorate 
very  rapidly,  rendering  a  difference  of  from  1  to  3  points  on  the 
scale  of  the  colorimeter, 

BIBLIOGRAPHY. 

Abderhalden:     Lehrbuch  der  biochemischen  Arbeitsmethoden,  vol.  ii,  p.  180. 

Agnew:     Arch.  Int.  Med.,  1914,  vol.  xiii,  p.  485. 

Allen:     Glycosuria  and  Diabetes,  1913,  Harvard  University  Press. 

Bang:    Der  Blutzucker,  Wiesbaden,  1913. 

Bang:     Biochem.  Ztschr.,  vol.  ii  and  xl. 

Bertrand:     Bull,  de  Soc.  chim.  de  France,  1906,  vol.  xxxvi,  p.  1285. 

Bierry:     Compt.  rend.  Acad.  d.  sc.,  1914,  vol.  clviii,  pp.  61-64;  Compt.  rend. 

Soe.  de  biol.,  1914,  vol.  Ixxvi,  pp.  261,  386-388. 
Boe:      Biochem.  Ztsehr.,   1913,  vol.   Iviii,   pp.   106-118. 
Chelle :    Compt.  rend.  Soc.  de  biol.,  1914,  vol.  Ixxvi,  pp.  852-855. 
Dorner:    Ztschr.  f.  klin.  Med.,  Berlin,  1914,  vol.  Ixxix,  pp.  287-295. 
Epstein:     Jour.  Am.  Med.  Assn.,  1914,  vol.  Ixiii,  p.  1667. 
Fandern:     Compt.  rend.  Soc.  de  biol.,  1914,  vol.  Ixxvi,  pp.  68-70. 
Farr  and  Austin:     Jour.  Exper.  Med.,  1913,  vol.  xviii,  p.  288. 
Farr  and  Krumbhaar:     Jour.  Am.  Med.  Assn.,  1914,  vol.  Ixiii,  p.  2214. 
Flatow:    Deutsch.  Arch.  f.  klin.  Med.,  vol.  cv,  p.  58. 
Folin:      Jour.   Biol.  Chem.,   1915,  vol.  xxii,  p.   327. 
Folin  and  Denis:     Jour.  Biol.  Chem.,  1913,  vol.  xiv,  p.  29;   Ibid.,  1914,  vol. 

xvii,  p.  487. 

Folin,  Denis,  and  Seymour :     Arch.  Int.  Med.,  1914,  vol.  xiii,  p.  224. 
Folin,  Karsner,  and  Denis :    Jour.  Exper.  Med.,  1912,  vol.  xvi,  p.  789. 
Frank:     Ztschr.  f.  physiol.  Chem.,  vol.  Ixx. 
Frank:     Deutsch.  Arch.  f.  klin.  Med.,  vol.  ciii. 
Frank  and  Isaak:    Ztschr.  f.  exper.  Path.  u.  Therap.,  1909,  vol.  vii. 
Frothingham,  Fitz,  Folin  and  Denis:     Arch.  Int.  Med.,  1913,  vol.  xii,  p.  245. 
Frothingham  and  Smillie:      Arch.   Int.   Med.,  1914,  vol.  xiv,   p.  541. 
Gardner   and   McLean:      Biochem.  Jour.,   1914,   vol.   viii,   p.   391. 
Gilbert:      Semaine  med.,  1909. 

Griesbau:     Ztschr.  f.  physiol.  Chem.,  1913,  vol.  Ixxxviii,  pp.  199-209. 
Hagelberg:      Berl.  klin.   Wchnschr.,   1912,   No.  40. 
Hawk:     Physiological  Chemistry. 

Hopkins :     Am.  Jour.  Med.  Sc.,  1915,  vol.  cxliv,  p.  254. 
Joslin,  E.  P.:     Diabetes,  1916. 

Karsner  and  Denis:     Jour.  Exper.  Med.,  1914,  vol.  xix,  pp.  259  and  270. 
Kumagava    Suto,    Salkowski    Festschrift,    1904. 
Lewis  and  Benedict:      Jour.  Biol.  Chem.,  1915,  vol.  xx,  p.   61. 
Liefmann  and  Stern:     Biochem.  Ztschr.,  vol.  i,  p.  299. 

Macleod:    Diabetes,  Its  Pathological  Physiology,  London  and  New  York,  1913. 
Maguitz :     Montsf  t.  f .  kinderh,  1914,  vol.  xii,  pp.  569-585. 
Marshall  and  Davis:     Jour.  Biol.  Chem.,  1914,  vol.  xviii,  p.  53. 
McLean  and  Selling:     Jour.  Biol.  Chem.,  1914,  vol.  xix,  p.  31. 
Michaelis  and  Rona:    Biochem.  Ztschr.,  1908,  vols.  vii  and  xiv. 
Moeckel  and  Frank:     Ztschr.  f.  physiol.  Chem.,  vol.  Ixv,  p.   323;   Ibid.,  vol. 

Ixix,  p.  84;   bach  and  Severin,  Zentralbl.  f.  d.  ges.  Physiol.  u.  Path.  d. 

Stoffwechs.,   1911,  pp.   55,   177,   and   665. 

Muller:     Ztschr.  f.  physiol.  Chem.,  1914,  vol.  xci,  pp.  287-291. 
Myers  and  Baily :     Jour.  Biol.  Chem.,  1916,  vol.  xxiv,  p.  147. 
Myers  and  Fine:     Jour.  Biol.  Chem.,  1915,  vol.  xx. 
Myers  and  Fine:     Essentials  of  Pathological  Chemistry. 


SUGAR    IN    BLOOD  33 

Xaunyn:     Der  Diabetes  Militus,  Wien,  1906,  p.  37. 

Neubauer:      Bioehem.   Ztschr.,  vol.   xxv. 

Pavy:     Lancet,  London,  p.  4269. 

Pearce:     Jour.  Biol.  Chem.,  1915,  vol.  xxii,  p.  525. 

Eeicher  and   Stein:      Bioehem.  Ztschr.,  vol.  xxxvii.      Tr.   Kongress  f.  innere 

Medizin,  Wiesbaden,  1910. 

Roily   and   Oppermann:      Bioehem.   Ztschr.,  vols.   xxxviii   and   xxxix. 
Schirokauer :     Berl.  klin.  Wchnschr.,  1912,  No.   38,  p.   1783;    Ibid.,  1911,  p. 

1505. 

Scott:     Am.  Jour.  Physiol.,  1914,  vol.  xxxiv,  pp.  271-311. 
Shaffer:     Jour.  Biol.  Chem.,  1914,  vol.  xix,  pp.  285-295,  and  297-302. 
Steustrom:     Bioehem.  Ztschr.,  1914,  vol.  Iviii,  pp.  472-482. 
Stilling :     Arch,  f .  exper.  Path.  u.  Therap.,  vol.  Ixvi,  p.  238. 
Strouse:     The  Accurate  Clinical  Study  of  Blood  Sugar,  Bull.  Johns  Hopkins 

Hosp.,  1915. 

Tachu:     Arch.  f.  klin.  Med.,  1911,  vols.  cii  and  civ,  p.  437. 
Tachu:      Ebenda,  vol.  civ,  p.   437. 
Takataschi:     Bioehem.  Ztschr.,  vol.  xxxvii. 

Taylor  and  Hutton:      Jour.  Biol.  Chem.,  1915,  vol.  xxii,  p.  63. 
Tileston  and  Comfort:      Arch.  Int.  Med.,   1914,  vol.  xiv,  p.  620. 
von  Hess:      The   Condition   of   the   Sugar   in   the   Blood,   Jour.   Pharm.   and 

Exper.  Therap.,  1914. 

von  Noorden:     Die  Zuckerkrankheit,  Berlin,  1912,  p.  59. 
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Ixvi. 
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CHAPTER  III. 


CKEATININE. 

We  begin  this  estimation  by  taking  the  remaining  nitrate  as 
already  described  in  the  sugar  estimation,  i.e.,  that  part  of  the 
nitrate  left  after  we  took  the  3  c.c.  for  the  sugar  test.  Take  10 
c.c.  of  this  nitrate.  (At  this  point  we  wish  to  emphasize  the  fact 
that  in  this  test  the  unknown  and  the  standard  solution  must  be 
made  up 'at  the  same  time  to  prevent  the  development  of  the  color 
in  one  case  faster  than  that  in  the  other,  thereby  obtaining  in- 
correct results.)  To  the  10  c.c.  nitrate  add  0.5  c.c.  of  a  10%  sodium 
hydroxide  solution,  and  to  20  c.c.  of  the  standard  creatinine  solu- 
tion add  1  c.c.  of  10%  sodium  hydroxide.  (See  Plate  II  for  the 
color  of  the  standard  creatinine  wedge.)  Allow  both  to  stand  10 
minutes  and  read  in  the  colorimeter. 

TABLE  II1 

ESTIMATION  OF  CREATININE  IN  THE  BLOOD   WITH   THE   HELLIGE  COLORIMETER 


COLORI- 
METRIC 
READING 

CREATININE 
MGMS.  PER 
DILUTION 
OF  100  C.C. 

COLORI- 
METRIC 
READING 

CREATININE 
MGMS.  PER 
DILUTION 
OF  100  C.C. 

COLORI- 
METRIC 
READING 

CREATININE 
MGMS.  PER 
DILUTION 
OF  100  C.C. 

40 

0.80 

57 

0.55 

74 

0.31 

41 

0.78 

58 

0.54 

75 

0.30 

42 

0.77 

59 

0.52 

76 

0.28 

43 

0.75 

60 

0.51 

77 

0.27 

44 

0.74 

61 

0.50 

78 

0.25 

45 

0.72 

62 

0.48 

79 

0.24 

46 

0.71 

63 

0.47 

80 

0.22 

47 

0.70 

64 

0.45 

81 

0.21 

48 

0.68 

65 

0.44 

82 

0.20 

49 

0.67 

66 

0.42 

83 

0.18 

50 

0.65 

67 

0.41 

84 

0.17 

51 

0.64 

68 

0.40 

85 

0.15 

52 

0.62 

69 

0.38 

86 

0.14 

53 

0.61 

70 

0.37 

87 

0.12 

54 

0.60 

71 

0.35 

88 

0.11 

55 

0.58 

72 

0.34 

89 

0.10 

56 

0.57 

73 

0.32 

90 

0.09 

^he  table  here  given  must  be  used  when  N/4  bichromate  is  used  as  a  standard.  From 
Meyers  and  Fine:  Chemical  Composition  of  the  Blood  in  Health  and  Disease,  New 
York,  1915. 


CREATININE  35 

The  standard  solution  of  creatinine  is  made  by  dissolving  15 
mgms.  of  pure  creatinine  in  1000  c.c.  of  a  saturated  solution  of 
picric  acid. 

The  formula  for  the  computation  of  this  result  is  as  follows: 
89  minus  reading  x  0.0179  x  5  =  mgms.  of  creatinine  per  100  c.c. 
of  blood. 

Example. — Let  us  assume  the  reading  in  an  experiment  is  64. 
Then  89  minus  64  =  25x0.0179  =  0.4475x5  =  2.2375  mgms.  (nor- 
mal). 

Slightly  less  accurate  results  than  these  may  be  obtained  by 
using  N/4  bichromate  of  potash  solution.  (See  Plate  II  for 
the  color  of  the  standard  bichromate  wedge.)  When  using  this 
solution  as  a  standard  the  nitrate  is  treated  as  in  the  preceding, 
and  the  result  is  multiplied  by  5.  The  reader  is  referred  to  Table 
II  (page  34)  which  should  be  used  when  N/4  potassium  bichromate 
is  used  as  a  standard. 

The  standard  potassium  bichromate  is  made  by  dissolving  12.28 
grams  of  potassium  bichromate  in  distilled  water  and  making  up 
to  1  liter. 

The  authors  recommend  the  pure  creatinine  over  the  latter 
method  inasmuch  as  repeated  .experiences  with  the  two  methods 
give  greater  percentages  of  accurate  findings  with  the  former. 

BIBLIOGRAPHY. 

Chace  and  Myers:     Jour.  Am.  Med.  Assn.,  1916,  vol.  Ixvii,  p.  932. 

Folin:     Jour.  Biol.  Chem.,  1914,  vol.  xvii,  p.  475. 

Folin  and  Denis:     Jour.  Biol.  Chem.,  1912,  vol.  xii,  p.  14 J;  Ibid.,  1914,  vol. 

xvii,  pp.  475,  487,  and  493. 
Foster:     Arch.  Int.  Med.,  1915,  vol.  xv,  p.  356. 
Myers   and  Fine:      Jour.   Biol.  Chem.,   1915,  vol.  xx,  p    391. 
Myers  and  Lough:     Arch.  Int.  Med.,  1915,  vol.  xvi,  p.  536. 
Woods:     Arch.  Int.  Med.,  1915,  vol.  xvi,  p.  577. 


'i_l 


CHAPTER  IV. 
CREATINE. 

For  the  determination  of  creatine  (and  creatinine),  pipette  with 
an  Ostwald-Folin  pipette  1  or  2  c.c.  of  the  remaining  filtrate 
from  the  sugar  estimation  into  a  small  test  tube  or  10  c.c.  graduate, 
and  autoclave  at  twenty  pounds  pressure  for  twenty  minutes.  At 
the  end  of  this  time  cool  the  solution,  make  up  to  8  c.c.  with  a 
saturated  solution  of  picric  acid,  and  then  add  0.4  c.c.  of  a  10% 
sodium  hydroxide  solution.  At  this  point  it  is  also  well  to  empha- 
size the  fact  that  the  unknown  and  the  standard  must  be  made  up 
at  the  same  time.  To  20  c.c.  of  standard  creatinine,1  add  1  c.c.  of  a 
10%  solution  of  sodium  hydroxide  (this  should  be  added  at  the 
same  time  the  0.4  c.c.  is  added  to  the  unknown)  and  then  compare 
the  unknown  and  the  standard  after  standing  for  ten  minutes. 
The  formula  for  computation  of  this  result  is  as  follows :  89  minus 
reading  x  0.0179  x  20  =  mgms.  creatinine  and  creatine. 

Slightly  less  accurate  results  may  be  obtained  by  using  N/4  po- 
tassium bichromate2  as  a  standard. 

If  the  accurate  value  of  creatine  is  desired,  this  is  obtained  by 
subtracting  the  value  of  creatinine  from  the  creatine  and  creatinine 
and  multiplying  it  by  1.16. 

Example. — Let  us  assume  that  the  reading  was  69.  Then  89 
minus  69  =  20  x  0.0179  =  0.358  x  20  =  7.16  mgms.  of  creatinine  +  cre- 
atine =  7.16  -  2.2375  (mgms.  creatinine)  =4.9225x1.16  =  5.7101 
mgms.  creatine  per  100  c.c.  blood  (normal). 


'This  standard  is  made  by  dissolving  15  mgms.  of  pure  creatinine  and  making  un  to 
one  liter  with  saturated  picric  acid. 

"This  is  prepared  by  dissolving  12.28  grams  of  potassium  bichromate  in  distilled  water 
and  making  up  to  1000  c.c. 


CHAPTER  V. 
URIC  ACID. 

Place  10  c.c.  of  blood  in  a  casserole  (Fig.  12)  of  at  least  375 
c.c.  capacity.  Add  50  c.c.  of  N/100  acetic  acid. 

The  N/100  acetic  acid  is  prepared  by  adding  0.6  c.c.  glacial 
acid  to  1  liter  of  distilled  water. 

This  lasts  about  two  weeks  and  should  be  cast  aside  after  that 
time  and  a  new  solution  made. 

Place  the  casserole  in  a  water-bath  and  heat  until  coagulation 
takes  place.  This  usually  takes  about  ten  minutes  with  an  cffi- 


Fig.  12.— Casserole. 

cient  water-bath.  Heat  the  casserole  over  a  free  flame  until  it 
comes  to  a  boil,  stirring  continuously.  Now  add  about  one  spoon- 
ful (4  c.c.)  of  alumina  cream.  (For  the  preparation  of  alumina, 
cream,  take  500  c.c.  of  8%  aluminum  acetate  in  acetic  acid.  This) 
8%~solution  may  be  purchased  from  any  reliable  chemical  house. 
Precipitate  this  with  sodium  bicarbonate  (dry)  until  the  solution 
is  neutral.  This  is  verified  by  litmus  paper  estimation.  Allow 
this  to  stand  24  hours  and  decant  the  supernatant  fluid.  This  is 
repeated  six  times,  that  is,  add  distilled  water  and  mix  and  allow 
to  stand  another  24  hours.  In  this  way,  it  takes  about  six  days 
to  make  this  reagent.  On  the  last  day  the  precipitate  is  filtered 
and  put  in  a  jar,  with  the  addition  of  5  c.c.  of  chloroform.  It  is 
now  ready  for  use.  It  should  be  kept  in  the  ice-box  for  storage.) 
Boil  for  one  minute,  stirring  continuously.  We  now  filter  this 
solution  and  wash  back  the  coagulum  on  the  filter  paper  into 


38  BLOOD   AND   URINE    CHEMISTRY 

the  casserole  with  about  100  c.c.  of  hot  distilled  water.  Heat  this 
mixture  in  the  casserole  over  a  free  flame  to  the  boiling  point,  and 
filter.  Evaporate  the  combined  filtrates  down  to  1  or  2  c.c.  in  the 
following  manner.  Boil  slowly  over  a  free  flame  until  the  volume 
has  been  reduced  to  about  50  c.c.  Continue  the  evaporation  in  the 
water-bath  down  to  1  or  2  c.c.  Transfer  this  to  a  conical  centri- 
fuge tube  of  15  c.c.  capacity,  washing  the  casserole  with  two  or  three 
hot  water  portions.  The  final  volume  in  the  centrifuge  tube  should 
be  kept  lelow  10  c.c.  When  this  has  cooled,  add  fifteen  drops  of 
ammoniacal-silver-magnesium1  mixture  and  the  tube  is  shaken  and 
placed  in  a  refrigerator  for  about  fifteen  minutes  (to  allow  for 


Fig.   13. — Showing  centrifuge   tube  attached  to   suction. 

the  precipitation  of  purine).  Centrifuge  the  tube  from  three  to 
five  minutes,  then  invert,  and  pour  off  the  supernatant  fluid. 
Wipe  the  lip  of  the  tube  with  filter  paper  and  allow  the  ammonia 
to  volatilize  by  suction.  This  is  accomplished  by  attaching  the  cen- 
trifuge tube  to  the  rubber  tubing  of  the  Chapman  pump  (Fig.  13). 
We  are  now  ready  for  the  development  of  color  and  the  read- 
ing. As  before  mentioned,  the  beginner  should  work  as  fast  as 
possible  as  the  color  may  fade  or  turbidity  may  develop.  It  is  a 
general  axiom,  of  course,  that  turbid  solutions  cannot  very  well 
be  read  in  a  colorimeter. 

JFor  the  preparation  of  ammoniacal-silver-magnesium  mixture,  mix  70  c.c.  of  3%  sil- 
ver nitrate  solution,  30  c.c.  of  magnesium  mixture,  and  100  c.c.  of  concentrated  ammonia. 
Any  turbidity  which  may  develop  is  removed  by  filtration.  The  magnesia  mixture  al- 
luded to  is  made  as  follows:  Dissolve  35  grams  of  magnesium  sulphate  and  70  grams  of 
ammonium  chloride  in  280  c.c.  of  distilled  water  and  then  add  140  c.c.  of  concentrated 
ammonia. 


URIC   ACID 


39 


Prepare  a  100  c.c.  graduated  cylinder  for  the  unknown  and  a 
50  c.c.  volumetric  flask  for  the  standard  solution  (Fig.  14).  Then 
pipette  5  c.c.  of  uric  acid  standard2  (5  c.c.=l  mgm.  of  uric  acid) 
into  the  50  c.c.  volumetric  flask.  To  the  uric  acid  standard  add  two 
drops  of  a  5%  solution  of  potassium  cyanide,  2  c.c.  of  Folin-Macal- 
lum3  reagent,  20  c.c.  of  saturated  sodium  carbonate,  and  in  one 
minute  add  water  to  the  50  c.c.  mark.  (See  Plate  I  for  the  color 
of  the  standard  uric  acid  wedge.)  To  the  precipitate  in  the  centri- 
fuge tube  add  2  drops  of  a  5%  potassium  cyanide  solution  (the  tube 


Fig.   14. — Volumetric  flask. 


is  shaken  so  as  to  dissolve  the  precipitate) ,  and  2  c.c.  of  the  Folin- 
Macallum  reagent,  and  then  wash  the  contents  of  the  centrifuge 
tube  into  a  100  c.c.  graduate  with  from  15  to  20  c.c.  of  saturated 
sodium  carbonate.  If  the  color  is  developed  well,  use  more  car- 
bonate, i.  e.,  20  c.c.  when  the  color  is  stronger  than  the  standard, 


2For  the   preparation   of  standard  uric   acid 
d  1 
ake 


lution,    dissolve   9    gms.    pure   crystalline 


ho 

solution  on  200 


n  disodi  urn  phosphate  and  1   gm.  dihyjdrogen  sodium  phosphate  in  200  to   300  c.c. 
illgd  wTUii'.     BiltiP'tfid  make  1  1  i.i^l  U    1  1  m  i  o  inr*¥fflT'ftTrt~rirnri^^**r  n  1  1  r  this  warm,  clear 


(Kahlbaum)   suspended  in  a  few  cubic  cen- 
iter voumetric  flask.     Agitate  until  completely  dissolved,  add  at 


timeters  of  warer 

once  exactly  1.4  c.c.  glacial  acetic  acid.  Make  up  to  one  liter,  mix  and  add  5  c.c.  chlo- 
roform. 5  c.c.  of  this  solution  are  equivalent  to  1  ingm.  of  uric  acid.  This  solution 
should  be  freshly  prepared  once  every  two  months.  Before  weighing  out  the  200  mgms. 
of  uric  acid  it  is  well  to  dry  over  night  the  quantity  from  which  the  measure  is  to 
be  made  in  a  drying  oven  at  100°  C. 

•For  the  preparation  of  Folin-Macallum  reagent,  boil  100  gms.  of  sodium  tungstate, 
20  c.c.  concentrated  hydrochloric  acid,  and  30  c.c.  of  85%  phosphoric  acid  in  750  c.c. 
distilled  water  for  two  hours  and  then  make  up  to  1000  c.c.  with  distilled  water.  In 
boiling,  it  is  well  to  have  a  funnel  over  the  flask  so  as  to  prevent  undue  evaporation. 


40 


BLOOD   AND   URINE    CHEMISTRY 


and  15  c.c.  when  it  is  weaker.  The  fundamental  principle  of  these 
dilutions  in  microchemical  work  is  to  have  the  unknown  solution 
weaker  in  color  than  the  standard  solution.  A  period  of  time  of 
from  forty  to  sixty  seconds  should  be  allowed  to  elapse  before  de- 
termining whether  to  dilute  to  50  or  100  c.c.  Dilute  with  dis- 
tilled water  to  25,  50,  or  100  c.c.,  depending  upon  the  depth  of/ 
color  obtained.  Table  III  gives  the  data  for  working  out  the 
amount  of  uric  acid  present. 

Example. — Suppose  the  final  dilution  of  the  unknown  was  25 
and  the  reading  was  42.  42  in  the  table  is  equivalent  to  1.24 
mgms.  This  is  divided  by  4  because  it  is  14  as  strong  as  the 
amount  in  the  table  (i.e.,  14  of  10°)  which  equals  0.31  mgms.  in 
10  e.c.  of  blood  (which  is  the  amount  of  blood  we  started  with). 
In  100  c.c.  of  blood  \ve  would  have  10  x  0.31=3.1  mgms. 


TABLE  III4 


ESTIMATION  OF  URIC  ACID  WITH  HELLIGE  COLORIMETER 


COLORI- 

URIC  ACID 

COLORI- 

URIC  ACID 

COLORI- 

VRIC   ACID 

METRIC 

MGMS.  PER 

METRIC 

MGMS.  PER 

METRIC 

MGMS.  PER 

READING 

DILUTION 

READING 

DILUTION 

READING 

DILUTION 

OF    100   C.C. 

OF    100   C.C. 

OF  100  C.C. 

20 

1.67 

40 

.28 

60 

0.88 

21 

1.65 

41 

.26 

61 

0.86 

22 

1.63 

42 

.24 

62 

0.84 

23 

.61 

43 

.22 

63 

0.82 

24 

.59 

44 

.20 

64 

0.81 

25 

.57 

45 

.18 

65 

0.79 

26 

.55 

46 

.16 

66 

0.77 

27 

.53 

47 

.14 

67 

0.75 

28 

.51 

48 

.12 

68 

0.73 

29 

.49 

49 

.10 

69 

0.71 

30 

1.48 

50 

1.08 

70 

0.69 

31 

1.46 

51 

1.06 

71 

0.67 

32 

1.44 

52 

1.04 

72 

0.65 

33 

1.42 

53 

1.02 

73 

0.63 

34 

1.40 

54 

1.00 

74 

0.61 

35 

1.38 

55 

0.98 

75 

0.59 

36 

1.36 

56 

0.96 

76 

0.57 

37 

1.34 

57 

0.94 

77 

0.55 

38 

1.32 

58 

0.92 

78 

0.53 

39 

1.30 

59 

0.90 

79 

0.51 

4Myers   and   Fine:      Chemical    Composition   of   the   Blood 


Health   and    Disease,    New 


URIC   ACID  41 

BIBLIOGRAPHY. 

Benedict  and  Hitchcock:     Jour.  Biol.  Chem.,  1915,  vol.  xx,  pp.  619,  629,  and 

633. 

Chace  and  Myers:     Jour.  Am.  Med.  Assn.,  1916,  vol.  Ixvii,  pp.  931,  932. 
Fine  and  Chace:     Jour.  Pharm.  and  Exper.  Therap.,  1914,  vol.  vi,  p.  219. 
Folin  and  Denis:     Arch.  Int.  Med.,  1915,  vol.  xvi,  p.  33;  Jour.  Biol.  Chem., 

1912,  vol.  xiii,  p.  469;  Ibid.,  1913,  vol.  xiv,  pp.  29  and  95. 
Folin  and  Macallum:     Jour.  Biol.  Chem.,  1912,  vol.  xiii,  p.  363. 
Myers  and  Fine:     "Blood  in  Health  and  Disease,"  1915,  p.  14. 
Weiss:     New  York  Med.  Jour.,  1914,  vol.  c,  p.  180. 


CHAPTER  VI. 
UREA. 

Into  a  test  tube  that  will  readily  slip  into  a  100  c.c.  graduated 
cylinder  introduce  2  c.c.  of  distilled  water  and  0.1  gin.  of  urease,1 
and  2  c.c.  of  blood  with  an  Ostwald  pipette ;  .  then  incubate 
the  tube  in  a  beaker  of  water  at  50°  C.  for  one-half  hour.  At  the 
end  of  this  time  add  two  drops  of  caprylic  alcohol  or  1  c.c.  of 
amylic  alcohol  to  prevent  foaming  in  aeration. 

We  now  direct  our  attention  to  the  manner  of  setting  up  the 
glassware  for  the  continuation  of  this  test.  The  chemistry  of 
this  estimation  is  about  as  follows:  The  enzyme  urease  converts 
urea  into  ammonium  carbonate.  The  ammonia  is  then  liberated 
by  aeration  in  the  presence  of  sodium  carbonate  in  excess  and 
goes  over  into  the  hydrochloric  acid  as  ammonium  chloride.  This 
can  be  determined  colorimetrically  by  the  use  of  Nessler's  reagent. 
There  should  be  two  cylinders  for  each  sample  of  blood.  If  more 
than  one  specimen  of  blood  is  to  be  examined,  these  cylinders  may 
be  run  in  series,  two  for  each  test.  One  cylinder  is  graduated,  the 
other  nongraduated.  Fig.  15  shows  the  manner  of  arranging  this 
glassware. 

A  two-hole  rubber  stopper  is  placed  in  each  cylinder.  Cylinder 
1  (A- A')  is  graduated  and  is  connected  with  the  suction.  Cyl- 
inder 2  (B-B')  is  nongraduated  and  is  connected  with  the  acid 
wash  (C)  bottle.  This  acid  wash  bottle  is  simply  a  bottle  con- 
taining sulphuric  acid  (10%)  placed  at  the  end  of  the  outfit  to  pre- 
vent the  ammonia  in  the  air  from  gaining  entrance  into  the  test. 
Cylinder  1  (A- A')  has  a  short  tube  bent  at  right  angles  connected 
to  the  suction  and  only  extending  in  the  cylinder  to  a  point  just 
within  the  cylinder.  This  is  tube  F-F'.  Tube  G-G'  extends  almost 
to  the  bottom  of  cylinder  1.  It  has  a  sealed  ending  with  small  holes 
punched  in  its  side.  This  can  readily  be  done  as  follows:  The 
holes  may  be  made  with  a  platinum  wire  which  is  at  white  heat, 
provided  the  glass  is  only  moderately  hot.  Cylinder  2  has  a  right- 
crease  may  be  purchased  from  the  Arlington  Chemical  Co.,  Yonkers,  N.  Y. 


UREA 


43 


44  BLOOD   AND   URINE    CHEMISTRY 

angle  tube  extending  to  a  point  just  below  the  stopper  (D).  It 
has  another  tube  with  a  straight  open  end  dipping  into  the  test 
tube  (E)  and  running  out  to  be  connected  either  with  the  acid 
wash  bottle  extension  or  with  another  series  of  cylinders  in  case 
more  than  one  specimen  of  blood  is  under  examination. 

Into  the  100  c.c.  graduated  cylinder  (cylinder  1)  place  20  c.c. 
distilled  water  and  two  to  three  drops  of  10%  hydrochloric  acid. 
Now  close  cylinder  1  and  open  cylinder  2.  To  the  test  tube  con- 
taining the  digested  blood  allow  an  equal  volume  of  saturated 
sodium  carbonate  to  slowly  run  down  under  the  blood.  Immedi- 
ately and  carefully  insert  the  tube  into  cylinder  2  and  immediately 
close,  and  then  carefully  and  tightly  seal  the  connection.  The  suc- 
tion is  started  by  means  of  the  Chapman  pump,  the  rate  is  slow 
for  about  five  minutes  and  then  gradually  increased  as  much  as 
the  apparatus  will  stand.  The  aeration  is  kept  up  from  thirty  to 
forty-five  minutes.  At  the  end  of  this  time,  disconnect  the  tube 
and  use  cylinder  1  for  the  final  determination.  Remove  the  rubber 
stopper  from  cylinder  1  and  wash  the  tube  with  distilled  water  (2 
to  3  c.c.). 

We  now  come  to  the  development  of  color.  Into  a  50  c.c.  volu- 
metric flask  pipette  5  c.c.  of  ammonium  sulphate  solution  contain- 
ing 1  mgm.  of  nitrogen  (this  is  the  standard  solution),  add  25  c.c. 
distilled  water,  and  then  20  c.c.  Nessler's  solution,  diluted  1  to  5. 
(See  Plate  I  for  the  standard  color  of  1  mgm.  of  nitrogen.) 

The  standard  ammonium  sulphate  solution  is  prepared  as 
follows : 

Dissolve  0.944  gm.  ammonium  sulphate  of  the  highest 
purity  in  distilled  water  and  make  up  to  1000  c.c.  in  a  volu- 
metric flask. 

Nessler  's  solution  is  prepared  as  follows :  for  one  liter  we  need : 

Mercuric  iodide  100  gins. 

Potassium  iodide  50  gms. 

Potassium   hydroxide  200  gms. 

Place  the  mercuric  iodide  and  the  potassium  iodide,  both  ' 
finely  powdered,  into  a  liter  volumetric  flask  and  add  about 
400  c.c.  distilled  water.  Now  dissolve  the  potassium 
hydroxide  in  500  c.c.  distilled  water,  cool  thoroughly,  and 
add  with  constant  shaking  to  the  mixture  in  the  flask.  Then 
make  up  to  one  liter  with  water.  This  usually  becomes  per- 
fectly clear.  Keep  at  37°  C.  in  incubator  over  night  or  until 


UREA 


45 


the  yellowish  white  precipitate  which  may  settle  out  is 
thoroughly  dissolved  and  only  a  small  amount  of  dark 
brownish  red  precipitate  remains.  The  solution  is  now 
ready  to  be  siphoned  off  and  used. 

To  cylinder  1  containing  the  unknown  in  the  form  of  ammonium 
chloride,  add  from  10  to  20  c.c.  of  diluted  Nessler's  solution  (1  to 
5),  dependent  upon  the  depth  of  color,  and  then  dilujte  tp  50  c.c.. 
100  c.c.,  etc.,  depending  upon  the  color.  The  colorimetric  reading 
should  be  made  at  once  and  computed  from  the  following  table  : 


TABLE  IV2 


ESTIMATION  OF  NITROGEN  WITH  THE  HELLIGE  COLORIMETER 


COLORI- 

NITROGEN 

COLORI- 

NITROGEN 

COLORI- 

NITROGEN 

METRIC 

MGMS.  PER 

METRIC 

MGMS.  PER 

METRIC 

MGMS.  PER 

READING 

DILUTION 

READING 

DILUTION 

READING 

DILUTION 

OF    100    C.C. 

OF    100    C.C. 

OF  100  C.C. 

20 

1.73 

40 

1.31 

60 

0.89 

21 

1.71 

41 

1.29 

61 

0.87 

22 

.69 

42 

1.27 

62 

0.85 

23 

.67 

43 

1.25 

63 

0.83 

24 

.65 

44 

1.23 

64 

0.81 

25 

.62 

45 

.20 

65 

0.78 

26 

.60 

46 

.18 

66 

0.76 

27 

.58 

47 

.16 

67 

0.74 

28 

.56 

48 

.14 

68 

0.72 

29 

.54 

49 

.12 

69 

0.70 

30 

.52 

50 

.10 

70 

0.67 

31 

.50 

51 

.08 

7* 

0.65 

32 

.48 

52 

.06 

72 

0.63 

33 

.46 

53 

.04 

73 

0.61 

34 

.44 

54 

.02 

74 

0.59 

35 

.41 

55 

0.99 

75 

0.56 

36 

.39 

56 

0.97 

76 

0.54 

37 

.37 

57 

0.95 

77 

•*  '0.52 

38 

.35 

58 

0.93 

78 

0.50 

39 

.33 

59 

0.91     - 

79 

0.48 

2Myers  and  Fine:  Chemical  Composition  of  the  Blood  in  Health  and  Disease,  New 
York,  1915. 

Example. — Suppose  the  dilution  was  to  50  and  our  reading  75. 
75  on  our  scale  is  equivalent  to  0.56  mgms.  Divide  this  by  2  be- 
cause our  dilution  was  to  50,  which  is  one-half  of  100,  which  will 
give  us  0.28  mgms.  in  2  c.c.  of  blood.  In  1  c.c.  of  blood  we  would 


46  BLOOD   AND   URINE    CHEMISTRY 

have  0.14  mgms.  of  urea  nitrogen  and  in  100  c.c.  of  blood  we  would 
have  14  mgms.,  which  is  about  normal. 

Should  it  be  desired  to  convert  this  urea  nitrogen  into  urea,  the 
results  are  always  multiplied  by  the  factor  2.14. 

BIBLIOGEAPHY. 

Chace  and  Myers:    Jour.  Am.  Med.  Assn.,  1916,  vol.  Ixvii,  pp.  931,  932. 
Combe  and  Levi:     Eev.  me"d.  de  la  Suisse  romande.,  1915,  vol.  xxxv,  p.  413. 
Folin  and  Denis:     Jour.  Biol.  Chem.,  1916,  vol.  xxvi,  p.  505;  Ibid.,  1912,  vol. 

xi,  p.  527. 

Folin  and  Pettibone:     Jour.  Biol.  Chem.,  1912,  vol.  xi,  p.  507. 
Foster:    Jour.  Am.  Med.  Assn.,  1916,  vol.  Ixvii,  p.  927. 
Kristeller:     Ztsehr.  f.  exper.  Path.  u.  Therap.,  1914,  vol.  xvi,  p.  496. 
Marshall:     Jour.  Biol.  Chem.,  1913,  vol.  xiv,  p.  283;  Ibid.,  1913,  vol.  xv,  pp. 

287  and  495. 

Neumann:     Biochem.  Ztsehr.,  1915,  vol.  Ixix,  p.  134. 
Olivieri:     Eiv.  osped.,  1914,  vol.  iv,  p.  221. 
Eose  and  Coleman:    Biochem.  Bull.,  1914,  vol.  iii,  p.  411. 
Siebeck:    Deutsch.  Arch.  f.  Min.  Med.,  1914,  vol.  cxvi,  p.  58. 
Van  Slyke  and  Cullen:     Jour.  Am.  Med.  Assn.,  1914,  vol.  Ixii,  p.  1558;  Jour. 

Biol.  Chem.,  1914,  vol.  xix,  p.  211 ;  Ibid.,  1916,  vol.  xxiv,  p.  117. 


CHAPTER  VII. 

NONPROTEIN  NITROGEN. 

iTv'i 

In  a  50  c.c.  volumetric  flask  with  about  35  c.c.  of  2.5%  trichlor- 
acetic  acid,  add  5  c.c.  of  blood,  and  make  the  volume  up  to  50  c.c. 
with  2.5%  trichloracetic  acid.  Shake  the  flask  vigorously,  and 
at  the  end  of  30  minutes  (or  as  soon  after  as  convenient)  filter 
the  solution  through  a  dry  filter.  To  the  filtrate  add  about  two 
grams  of  kaolin,  and  shake  the  solution  vigorously^  After  allow- 
ing the  mixture  to  stand  for  a  few  minutes  (5  to  10),  filter  again.J 
The  filtrate  should  now  be  quite  colorless.  Pipette  10  c.c.  of  the 
filtrate  (the  equivalent  of  1  c.c.  of  blood)  into  a  test  tube  about 
200  mm.  long  and  of  a  sufficient  diameter  to  slip  into  a  100  c.c. 


Fig.  16. — Microburner. 

graduated  cylinder  (no  lip).  Then  add  one-tenth  to  three-tenths 
of  a  gram  of  potassium  sulphate,  a  drop  of  10%  copper  sulphate, 
and  1  c.c.  of  concentrated  sulphuric  acid  in  the  order  named 
(these  reagents  should  be  of  the  highest  purity).  This  is  then 
boiled  over  a  microburner  (Fig.  16),  at  first  gently,  until  a  dark 
brown  color  appears. 

At  this  point  it  might  be  well  to  call  the  attention  of  the  reader 
to  a  modification  of  this  test1  which  will  serve  for  blood  as  well 
as  urine  estimations,  and  which  will  serve  to  shorten  this  test 
about  ten  minutes.  Allow  the  solution  to  cool  and  add  a  drop 
of  peroxide  of  hydrogen.  If  the  mixture  does  not  clear,  heat 
gently  over  the  microburner.  Repeat  this  process  once  more  if 
the  mixture  is  not  perfectly  clear  (digested).  One  drop  of  perox- 
ide of  hydrogen  will  usually  suffice.  Now  allow  the  tube  to  cool 
for  a  few  minutes  and  then  add  about  5  or  6  c.c.  of  distilled  water. 


Kiradwohl  and   Blaivas:     Jour.   Am.   Med.  Assn.,   Sept.  9,   1916,  vol.   Ixvii,  p.   809. 


BLOOD   AND    URINE    CHEMISTRY 


As  a  means  of  removing  fumes,  the  suction  is  connected  by  a 
two-hole  stopper  to  a  large  bottle  containing  a  solution  of  sodium 
hydroxide  (Fig.  17).  The  short  tube  A,  bent  at  right  angles,  should 
be  connected  to  the  suction.  The  tube  B  should  be  attached  to  a 


removing  fumes  in  connection  with  nitrogen  determinatic 


NONPROTEIN   NITROGEN  49 

funnel  over  the  mouth  of  the  test  tube  D.  After  a  few  determi- 
nations have  been  made,  it  is  well  to  wash  the  funnel  to  remove 
any  acid  which  may  have  condensed  upon  it. 

Aeration  is  carried  out  exactly  in  the  manner  as  for  urea, 
only  that  saturated  sodium  hydroxide  is  used  instead  of  saturated 
sodium  carbonate.  The  same  table2  is  also  used  for  calculation 
and  the  results  obtained  for  1  c.c.  of  blood.  ,  _  3>  X 

BIBLIOGRAPHY. 

Agnew:     Arch.  Int.  Med.,  1914,  vol.  xiii,  p.  485. 

Austin  and  Miller:     Jour.  Am.  Med.  Assn.,  1914,  vol.  Ixiii,  p.  944. 

Bock  and  Benedict:     Jour.  Biol.  Chem.,  1915,  vol.  xx,  p.  47. 

Farr  and  Austin:     Jour.  Exper.  Med.,  1913,  vol.  xviii,  p.  228. 

Farr  and  Krumbhaar:     Jour.  Am.  Med.  Assn.,  1914,  vol.  Ixiii,  p.  2214. 

Farr  and  Williams:     Am.  Jour.  Obst.,  1914,  vol.  Ixx,  p.  614;  Am.  Jour.  Med. 

Sc.,  1914,  vol.  cxlvii,  p.  556. 
Fitz:     Arch.  Int.  Med.,  1915,  vol.  xv,  p.  524. 
Folin:     Jour.  Biol.  Chem.,  1915,  vol.  xxi,  p.  195. 
Folin  and  Denis:      Jour.   Biol.  Chem.,  1912,  vol.  xi,  pp.   87,   161,  503,  527; 

Ibid.,  1912,  vol.  xii,  p.  141,  253 ;  Ibid.,  1913,  vol.  xiv,  p.  29 ;  Ibid.,  1913, 

vol.  xvii,  p.  487,   493;    Ibid.,   1915,  vol.  xxii,  p.   321;    Ibid.,   1916,  vol. 

xxvi,  p.  491. 

Folin,  Denis  and  Seymour:     Arch.  Int.  Med.,  1914,  vol.  xiii,  p.  224. 
Folin  and  Farmer:     Jour.  Biol.  Chem.,  1912,  vol.  xi,  p.  493. 
Folin  and  Lyman:     Jour.  Biol.  Chem.,  1912,  vol.  xii,  p.  259. 
Foster:     Arch.  Int.  Med.,  1915,  vol.  xv,  p.  356;  Jour.  Am.  Med.  Assn.,  1916, 

vol.  Ixvii,  p.  927. 

Frothingham:     Am.  Jour.  Med.  Sc.,  1915,  vol.  cxlix,  p.  808. 
Frothingham  and  Smillie:     Arch.  Int.  Med.,  1914,  vol.  xiv,  p.  541. 
Gradwohl  and  Blaivas :     Jour.  Am.  Med.  Assn.,  Sept.  9,  1916,  vol.  Ixvii,  p.  809. 
Greenwald:     Jour.  Biol.  Chem.,  1915,  vol.  xxi,  p.  61. 
Gulick:     Jour.  Biol.  Chem.,  1914,  vol.  xviii,  p.  541. 
Harding  and  Wareneford:     Jour.  Biol.  Chem.,  1915,  vol.  xxi,  p.  69. 
Hertz:     Wien.  klin.  Wchnschr.,  1914,  vol.  xxvii,  p.  323. 
Hohlweg:     Med.  Klin.,  1915,  vol.  xi,  p.  331;  Mitt.  a.  d.  Grenzgeb.  d.  Med.  u. 

Chir.,  1915,  vol.  xxviii,  p.  459. 

Hopkins  and  Jones:     Arch.  Int.  Med.,  1915,  vol.  xv,  p.  964. 
Karsner  and  Denis :     Jour.  Exper.  Med.,  1914,  vol.  xix,  p.  259. 
Lowy:     Ztschr.  f.  physiol.  Chem.,  1912,  vol.  Ixxix,  p.   349. 
McLean  and  Selling:     Jour.  Biol.  Chem.,  1914,  vol.  xix,  p.  31. 
Michand:     Cor.-Bl.  f.  schweiz.  Aerzte,  1913,  vol.  xliii,  p.  1474. 
Mosenthal:     Arch.  Int.  Med.,  1914,  vol.  xiv,  p.  844. 
Myers  and  Fine:     Jour.  Biol.  Chem.,  1915,  vol.  xx,  p.  391. 
Pepper  and  Austin:    Jour.  Biol.  Chem.,  1915,  vol.  xxii,  p.  81. 
Plass:     Am.  Jour.  Obst.,  1915,  vol.  Ixxi,  p.  608. 
Pribram:     Zentralbl.  f.  inn.  Med.,  1914,  vol.  xxxv,  p.  153. 
Schlutz  and  Pettibone :     Am.  Jour.  Dis.  Child.,  1915,  vol.  x,  p.  206. 
Taylor  and  Hulton:     Jour.  Biol.  Chem.,  1915,  vol.  xxii,  p.  63. 
Taylor  and  Lewis:     Jour.  Biol.  Chem.,  1915,  vol.  xxii,  p.  71. 
Tileston  and  Comfort:     Arch.  Int.  Med.,  1914,  vol.  xiv,  p.  620;   Am.  Jour. 

Dis.  Child.,  1915,  vol.  x.  p.  278. 
Woods:     Arch.  Int.  Med.,  1915,  vol.  xvi,  p.  577. 

*See  Table  IV,  p.  45. 

,00  .       ^     p^<- 


CHAPTER  VIII. 
CHOLESTEROL.1 

Preparation  of  Sample. — Run  2  c.c.  of  whole  blood,  plasma,  or 
serum  slowly  (a  slow  stream  of  drops)  from  a  pipette  into  about 
75  c.c.  of  a  mixture  of  redistilled  alcohol  and  ether  (3  parts  al- 
cohol, 1  part  ether)  in  a  100  c.c.  graduated  flask.  Keep  the  con- 
tents of  the  flask  in  motion  during  the  process  so  that  there  is 
no  clumping  of  the  precipitated  material.  Raise  contents  of  the 
flask  to  boiling  by  immersion  in  a  water-bath  (with  constant  shak- 
ing to  avoid  superheating),  cool  to  room  temperature,  fill  to  the 
mark  with  alcohol-ether,  mix  and  filter.  The  filtered  liquid  if 
placed  in  a  tightly  stoppered  bottle  in  the  dark  will  keep  un- 
changed for  a  considerable  time  so  that,  if  it  is  not  convenient 
to  complete  the  determination  at  once,  the  sample  may  be  carried 
to  the  above  stage  and  left  to  a  more  suitable  time. 

By  running  the  blood  slowly  into  the  large  quantity  of  alcohol- 
ether,  as  above,  the  protein  material  is  precipitated  in  finely  di- 
vided form  and  under  these  conditions  the  short  heating  combined 
with  the  great  excess  of  solvent  is  adequate  for  complete  extrac- 
tion of  serum  or  plasma.  The  extraction,  while  not  so  complete 
in  the  case  of  whole  blood,  is  believed  to  be  better,  because  of  the 
higher  values  obtained  than  that  obtained  by  any  other  method  in 
'  use  at  the  present  time. 

Determination. — Measure  10  c.c.  of  the  alcohol-ether  extract  in- 
to a  small  flat-bottomed  beaker  and  evaporate  just  to  dryness  over 
a  water-bath  or  electric  stove.  Any  heating,  after  dryness  is 
reached,  produces  a  brownish  color  which  passes  into  the  chloro- 
form and  renders  the  subsequent  determination  difficult  or  im- 
possible. The  cholesterol  is  extracted2  from  the  dry  residue  by 
boiling  out  three  or  four  times  with  successive  small  portions  of 
chloroform  and  decanting  into  a  10  c.c.  glass  stoppered,  gradu- 

^loor:     Jour.  Biol.   Chem.,  1916,  vol.  xxiv,  p.  229. 

2In  order  to  get  an  adequate  extraction  with  the  small  amounts  of  chloroform  used, 
an  excess  (3  c.c.)  should  be  added  each  time  and  the  mixture  allowed  to  boil  down  to 
half  its  volume  or  less,  before  decanting. 


CHOLESTEROL 


51 


ated  cylinder.  The  combined  extracts  after  cooling  (5  c.c.  or  less) 
are  then  made  up  to  5  c.c.  The  solution  should  be  colorless  but 
not  necessarily  clear,  since  the  slight  turbidity  clears  up  on  adding 
the  reagents. 

To  this  solution  add  2  c.c.  of  acetic  anhydride  and  0.1  c.c.  of 
concentrated  sulphuric  acid  and  after  mixing  place  in  the  dark 

TABLE  V3 
ESTIMATION  OF  CHOLESTEROL  WITH  THE  HELLIGE  COLORIMETER 


COLORI- 

CHOLESTEROL 

COLORI- 

CHOLESTEROL 

COLORI- 

CHOLESTEROL 

METRIC 

MGMS. 

METRIC 

MGMS. 

METRIC 

MGMS. 

READING 

DILUTION 

READING 

DILUTION 

READING 

DILUTION 

OF  5  C.C. 

OF  5  C.C. 

OP  5  C.C. 

15 

0.74 

35 

0.57 

55 

0.40 

16 

0.73 

36 

0.56 

56 

0.40 

17 

0.72 

37 

0.55 

57 

0.39 

18 

0.71 

38 

0.55 

58 

0.38 

19 

0.70 

39 

0.54 

59 

0.37 

20 

0.69 

40 

0.53 

60 

0.36 

21 

0.69 

41 

0.52 

61 

0.35 

22 

0.68 

42 

0.51 

62 

0.35 

23 

0.67 

43 

0.5) 

63 

0.34 

24 

0.66 

44 

0.50 

64 

0.33 

25 

0.65 

45 

0.49 

65 

0.32 

26 

0.65 

46 

0.48 

66 

0.31 

27 

0.64 

47 

0.47 

67 

0.30 

28 

0.63 

48 

0.46 

68 

0.30 

29 

0.62 

49 

0.45 

69 

0.29 

30 

0.61 

50 

0.45 

70 

0.28 

31 

0.60 

51 

0.44 

71 

0.27 

32 

0.59 

52 

0.43 

72 

0.26 

33 

0.59 

53 

0.42 

73 

0.25 

34 

0.58 

54 

0.41 

74 

0.24 

"This  table  is  good  for  both  standards  given  above  (cholesterol  and  Naphthol  Green 
B).  Myers  and  Fine:  Chemical  Composition  of  the  Blood  in  Health  and  Disease,  New 
York,  1915. 

for  10  minutes  to  allow  for  the  development  of  the  color..  Then 
compare  in  the  colorimeter  (Hellige)  with  a  standard  choles- 
terol solution  upon  which  the  color  is  developed  in  the  same  way.4 
See  Plate  I  for  the  standard  color  of  cholesterol. 


<For  the  preparation  of  standard  with  pure  cholesterol,  pipette  2  c.c.  of  an  0.08% 
freshly  prepared  chloroform  solution  of  cholesterol  into  a  dry,  accurately  graduated 
25  c.c.  cylinder  and  make  up  to  10  c.c.  with  chloroform  and  add  4  c.c.  acetic  anhydride 
and  0.2  c.c.  of  concentrated  sulphuric  acid.  Care  should  be  taken  that  the  unknown 
and  the  standard  are  made  together  and  both  the  colors  should  be  allowed  _  to  develop 
at  the  same  time.  The  reason  for  this  is  that  the  colors  fade  rather  rapidly.  It  is 
very  important  that  the  wedge  and  the  cup  of  the  colorimeter  be  perfectly  dry. 


52  BLOOD   AND   URINE    CHEMISTRY 

An  aqueous  solution  of  Naphthol  Green  B5  can  also  be  used  as 
a  standard.  The  cholesterol  in  0.2  c.c.  of  blood,  serum,  or  plasma, 
can  be  obtained  from  Table  V.  This  table  is  suitable  for  both  stand- 
ards (pure  cholesterol  or  Naphthol  Green  B). 

The  result  multiplied  by  500  will  give  the  percentage  of  choles- 
terol. 

Example.— Beading  is  60  which  equals  0.36  mgms.  cholesterol 
in  0.2  c.c.  blood,  plasma,  or  serum.  0.36  x  500=180  mgms.  or  0.18%. 

BIBLIOGRAPHY. 

Autenrieth  and  Funk:     Miinchen.  med.  Wchnschr.,  1913,  vol.  Ix,  p.  1243. 

Bang:     Chemie  und  Biochemie  der  Lipoide,  Wiesbaden,  1911,  pp.  20-27. 

Bloor:     Jour.  Biol.  Chem.,  1915,  vol.  xxiii,  p.  317;  Ibid.,  1916,  vol.  xxiv,  p.  227. 

Frank:     Jour.  Biol.  Chem.,  1916,  vol.  xxiv,  p.  431. 

Grigaut:    Compt.  rend.  Soc.  de  biol.,  1911,  vol.  Ixxi,  p.  531. 

Hanes:     Bull.  Johns  Hopkins  Hosp.,  1912,  vol.  xiii,  p.  77. 

Henes:     New  York  State  Jour.  Med.,  1915,  vol.  xv,  p.  310;  Jour.  Am.  Med. 

Assn.,  1914,  vol.  Ixiii,  p.   146. 
Lifschutz:     Ztsehr.  f.  physiol.  Chem.,  1907,  vol.  i,  p.  437;   Ibid.,  1907,  vol. 

liii,  p.  140 ;  Ibid.,  1908,  vol.  Iviii,  p.  175 ;  Ibid.,  1909,  Ixiii,  p.  223 ;  Ibid., 

1914,  vol.  xci,  p.  309;   Ibid.,  1914,  vol.  xcii,  p.   383;    Ibid.,   1914,  vol. 

xciii,  p.  209;  Biochem.  Ztsehr.,  1913,  vol.  Hi,  p.  206. 
Myers  and  Gorham:     Post-Graduate  Med.  Jour.,  1914,  vol.  xxix,  p.  938. 
Schmidt :    Arch.  Int.  Med.,  1914,  vol.  xii,  p.  123. 
Weston:     Jour.  Med.  Research,  1912,  vol.  xxvi,  p.  47. 
Weston  and  Kent:     Jour.  Med.  Research,  1912,  vol.  xxvi,  p.  531. 
Windaus :    Ztsehr.  f .  physiol.  Chem.,  1910,  vol.  Ixv,  p.  110. 

BFpr  the  preparation  of  Naphthol  Green  B,  dilute  2  c.c.  of  a  0.1%  aqueous  solu- 
tion "of  the  dye  to  17  c.c.  with  distilled  water.  The  diluted  solution  appears  to  keep 
for  a  little  time,  while  the  concentrated  solution  apparently  will  keep  for  a  considerable 
time.  The  permanency  of  the  solution  and  the  fact  that  the  color  is  practically  iden- 
tical with  that  obtained  from  cholesterol  makes  the  standard  very  convenient.  Myers 
and  Fine  have  found  this  solution  nearly  identical  with  the  pure  cholesterol  standard. 
They  advise,  however,  that  in  preparing  a  new  solution  it  is  best  to  standardize  it  by 
plotting  a  new  curve. 


CHAPTER  IX. 
TOTAL  SOLIDS. 

For  the  determination  of  total  solids,  a  weighing  bottle  with  a 
glass  stopper  and  a  glass  loop  (Fig.  18),  which  goes  inside  of 
the  bottle  when  stoppered,  to  which  a  block  of  filter  paper  is  fas- 
tened, is  required.1  From  an  accurately  graduated  pipette,  allow 
0.3-0.6  gms.  of  blood  to  flow  rapidly  on  the  filter  paper.  Quickly 
insert  the  stopper  to  prevent  any  loss  of  moisture,  weigh  the 
bottle.  Tilt  the  stopper,  and  then  place  the  bottle  in  a  drying 


Fig.    18. — Weighing  bottle   for   total   solids. 

oven  at  105°  C.  overnight.  Whenever  convenient,  the  bottle  is 
cooled  in  the  desiccator  (care  being  taken  that  the  stopper  is 
closed)  and  again  weighed.  From  the  loss  of  moisture  the  total 
solids  may  be  calculated. 

Calculation. — Divide  the  weight  of  the  residue  by  the  weight 
of  the  blood  used.  The  quotient  is  the  percentage  of  solids  con- 
tained in  the  blood  examined. 


'Myers   and   Fine:      Chemical    Composition    of   the    Blood    in   Health   and    Disease,    New 
York,   1915. 


CHAPTER  X. 
TOTAL  NITROGEN. 

Place  exactly  1  c.c.  of  blood  in  a  long-necked  Jena  glass  Kjel- 
dahl  flask  (Fig.  19),  add  20  c.c.  of  concentrated  sulphuric  acid  and 
about  0.2  grams  of  copper  sulphate,  and  boil  the  mixture  in  the 


Fig.  19.— Kjeldahl  flask. 

digestion  rack  (Fig.  20)  for  some  time  after  it  is  colorless  (about 
one  hour).  Allow  the  flask  to  cool  and  dilute  the  contents  with 
about  200  c.c.  of  ammonia-free  water.  Add  a  little  more  of  a 
saturated  sodium  hydroxide  solution  than  is  necessary  to  neutral- 
ize the  sulphuric  acid  (about  40  c.c.).  Introduce  into  the  flask 
a  little  coarse  pumice  stone  or  a  few  pieces  of  granulated  zinc 


TOTAL    NITROGEN 


55 


to  prevent  lumping,  and  a  small  piece  of  paraffin  to  lessen  the 
tendency  to  froth.  By  means  of  a  safety  tube  connect  the  flask 
with  a  condenser  (Fig.  21)  so  arranged  that  the  delivery  tube 
passes  into  a  vessel  containing  a  known  volume  (the  volume  used 


Fig.  20.— Digestion  rack. 


depending  upon  the  nitrogen  contents  of  the  blood)  of  N/10  sul- 
phuric acid  to  which  has  been  added  a  few  drops  of  congo  red,1 
care  being  taken  that  the  end  of  the  delivery  tube  reaches  be- 
neath the  surface  of  the  fluid.  This  delivery  tube  should  be  of  a 


Fig.  21. — Kjeldahl  apparatus  showing  condenser. 

large  caliber  in  order  to  avoid  the  sucking  back  of  the  fluid.  Mix 
the  contents  of  the  distillation  flask  very  thoroughly  by  shaking 
(or  rotating)  and  distil  the  mixture  until  about  two-thirds  of  the 
solution  has  passed  over.  Titrate  the  partly  neutralized  N/10 


J0.5   gm.   of  congo   red   in  a   mixture   of  90   c.c.    of  distilled  water   and    10   c.c.   of   95% 
ilcohol. 


56  BLOOD   AND   URINE    CHEMISTRY 

sulphuric  acid  against  N/10  sodium  hydroxide.2  Calculate  the 
amount  of  nitrogen  in  1  c.c.  of  blood  and  multiply  by  100  to  re- 
port for  100  c.c.  of  blood. 

Calculation. — 1  c.c.  of  N/10  sulphuric  acid  is  the  equivalent  of 
0.0014  gm.  nitrogen.  (Preparation  of  N/10  NaOH  and  N/10 
H2S04.) 

Folin-Farmer  Microchemical  Method. 

Pipette  exactly  1  c.c.  of  the  blood  into  a  25  c.c.  volumetric  flask. 
Then  dilute  with  distilled  water  up  to  25  c.c.  Now  pipette  1  c.c. 
of  the  diluted  blood  into  a  test  tube  of  such  a  size  that  it  will  slip 
into  the  aeration  apparatus  (Fig.  15).  Add  one  to  three-tenths  of 
a  gram  of  potassium  sulphate,  a  drop  of  10%  copper  sulphate 
solution,  and  1  c.c.  of  concentrated  sulphuric  acid  in  the  order 
named,  and  carry  out  digestion  as  in  the  determination  of  non- 
protein  nitrogen.  (See  page  47.)  The  result  obtained  above  is 
for  1/25  c.c.  of  blood. 

BIBLIOGRAPHY. 

Dakin  and  Dudly:     Jour.  Biol.  Chem.,  1914,  vol.  xvii,  p.  275. 
Folin  and  Denis:     Jour.  Biol.  Chem.,  1916,  vol.  xxvi,  p.  473. 
Folin  and  Farmer :     Jour.  Biol.  Chem.,  1912,  vol.  xi,  p.  493. 
Gulick:     Jour.  Biol.  Chem.,  1914,  vol.  xviii,  p.  541. 

Myers  and  Fine:     Post-Graduate,   1914-15;    reprinted  as   "Chemical  Compo- 
sition of  the  Blood  in  Health  and  Disease,"  New  York,  1915. 

2For  the  preparation  of  N/10  sodium  hydroxide,  dissolve  4  gms.  of  sodium  hydroxide 
in  about  900  c.c.  of  distilled  water.  Titrate  this  against  a  decinormal  solution  of  oxalic 
acid  which  is  made  by  dissolving  exactly  6.285  gms.  of  pure  oxalic  acid  in  a  liter  of 
distilled  water.  The  decinormal  sodium  hydroxide  was  purposely  made  too  strong; 
therefore,  less  than  10  c.c.  of  the  alkali  will  be  required  to  neutralize  10  c.c.  of  the 
decinormal  oxalic  acid  solution.  Suppose  that  9.5  c.c.  of  the  alkali  only  were  required, 
then  every  remaining  portion  of  9.5  c.c.  of  the  unknown  would  have  to  be  diluted  with 
0.5  c.c.  of  distilled  water.  This  solution  will  contain  the  equivalent  of  one-tenth  of  its 
molecular  weight  in  grams  (4  grams)  in  1000  c.c.  of  distilled  water.  From  this  N/10 
alkali,  N/10  HC1  may  be  prepared. 


CHAPTER  XL 


CHLORIDES.1 

Pipette  3  c.c.  of  blood  into  a  50  c.c.  graduated  centrifuge  tube 
(Fig.  22),  then  add  15  c.c.  of  N/100  acetic  acid  and  dilute  the 
volume  to  30  c.c.  with  distilled  water.  Place  the  tube  in  a  beaker 
of  boiling  water  to  bring  about  the  coagulation  of  the  protein, 
care  being  taken  that  the  contents  of  the  tube  are  agitated  oc- 
casionally with  a  stirring  rod.  After  the  protein  has  coagulated, 
the  tube  is  cooled,  again  made  to  volume  (30  c.c.),  and  centri- 
fuged.  After  this  is  done,  pour  the  slightly  col- 
ored supernatant  fluid  into  a  dry  centrifuge  tube 
and  add  about  six  drops  of  a  strong  solution  of 
colloidal  iron  and  place  the  tube  in  a  beaker  of 
hot  water  for  a  few  minutes.  This  brings  about  a 
complete  precipitation  of  all  protein.  After  cen- 
trifuging  (or  filtering)  the  clear  fluid  once  more, 
pour  it  from  the  tube  and  take  10  c.c.  (equavalent 
of  1  c.c.  of  blood)  into  a  50  c.c.  evaporating  dish 
or  a  25  c.c.  volumetric  flask,  depending  on  the 
method  used,  and  titrate. 

"Theoretically  the  Volhard-  Arnold  is  to  be  pre- 
ferred, but  the  substances  which  may  interfere 
with  the  Mohr  titration  are  so  small  that  the  re- 
sults are  practically  identical.  The  former  method 
advantage,  however,  when  for  any  reason  the 
fluid  to  be  titrated  has  been  rendered  acid." 

Volhard-Arnold  Method. 

Pipette  10  c.c.  of  the  filtrate  into  a  25  c.c.  volumetric  flask. 
Add  10  c.c.  of  the  standard  silver  nitrate  solution2  (1  c.c.  =  0.001 
gin.  of  sodium  chloride)  and  1  c.c.  of  the  ferric  alum  indicator,3 


JMyers  and  Fine:  Chemical  Composition  of  the  Blood  in  Health  and  Disease,  New 
York,  1915. 

-This  standard  is  prepared  by  dissolving  2.906  gms.  of  silver  nitrate  in  distilled  water 
and  making  up  to  1  liter. 

"The  indicator  is  made  by  dissolving  100  gms.  of  crystalline  ferric  ammonium  sulphate 
in  100  c.c.  of  25%  nitric  acid. 


58  BLOOD   AND   URINE    CHEMISTRY 

and  finally  make  up  to  volume  and  shake  thoroughly.  Cen- 
trifuge this  in  a  large  (50  c.c.)  centrifuge  tube  and  decant  the 
clear  supernatant  fluid.  Titrate  20  c.c.  of  the  fluid,  which  is  the 
equivalent*  of  0.8  c.c.  of  blood,  with  a  standard  ammonium  thio- 
cyanate  solution  of  the  same  strength  as  the  silver  nitrate,  until  a 
distinct  yellow  color  shows  throughout  the  mixture.  The  titration 
result,  divided  by  0.8,  subtracted  from  10,  to  obtain  the  silver 
nitrate  used,  and  multiplied  by  .001,  and  again  multiplied  by  100 
gives  the  percentage  of  chlorides  as  sodium  chloride. 

Example. — Beading  on  burette  is  3.2  c.c.  Divide  by  0.8  =  4; 
subtract  from  10  =  6;  multiply  by  0.001  =  0.006  (gms.  of  NaCl  in 
1  c.c.  of  blood)  ;  multiply  by  100  =  0.6%  (normal). 

Mohr  Method. 

Pipette  10  c.c.  of  the  filtrate  into  an  evaporating  dish  of  50 
c.c.  capacity  and  add  one  drop  of  a  10%  solution  of  potassium 
chromate.  Now  run  the  standard  silver  nitrate  (same  as  above,  1 
c.c.  equals  0.001  gm.  of  sodium  chloride)  into  the  dish  from  a 
burette  until  the  first  permanent  precipitate  of  silver  chromate, 
which  is  an  orange-red  color,  shows  throughout  the  whole  solu- 
tion on  stirring.  This  is  the  end  of  the  titration,  for  which  there 
is  a  correction  of  0.2  to  0.3  of  1  c.c.  This  result  multiplied  by 
0.001,  multiplied  by  100  gives  the  percentage  of  chlorides  as  so- 
dium chloride. 

Example. — Reading  on  burette  is  6.3  c.c.  Subtract  0.3  c.c., 
equals  6  (corrected  reading) ;  multiply  by  0.001  equals  0.006  (gms. 
of  NaCl  in  1  c.c.  of  blood)  ;  multiply  by  100  equals  0.6%  (normal). 

«Standard  ammonium  thiocyanate  is  prepared  by  dissolving  1.3  gms.  of  ammonium 
thiocyanate  in  800  c.c.  of  water,  titrating  against  the  above  silver  nitrate  standard,  and 
ascertaining  the  amount  of  water  which  must  be  added  to  the  solution  to  make  it  equiv- 
alent to  1  c.c.  of  the  standard  silver  nitrate  solution  or  0.001  gm.  of  sodium  chloride. 


CHAPTER  XII. 
TEST  FOR  ACIDOSIS  IN  BLOOD. 

Van  Slyke  Method  for  the  Determination  of  the  Carbon  Dioxide 
Combining  Power  of  Blood  Plasma. 

Having  centrifuged  the  fresh  oxalated  blood,  pipette  off  the  clear 
plasma  and  place  in  a  separatory  funnel  of  about  300  c.c.  capac- 
ity. Slight  heraolysis  does  not  affect  results  appreciably,  but 
hemolysis  should  be  avoided  as  much  as  possible  by  immediate 
centrifugalization.  In  order  to  determine  its  alkaline  reserve,  sat- 
urate the  plasma  with  carbon  dioxide  at  alveolar  tension.  In 
other  words,  the  operator  blows  vigorously  through  a  bottle  con- 
taining glass  beads  into  the  separatory  funnel,  as  shown  in  Fig. 
23.  If  one  blows  directly  into  the  separatory  funnel,  enough  mois- 
ture condenses  on  the  walls  of  the  funnel  to  appreciably  dilute 
the  plasma.  Close  the  funnel  at  stop-cock  S  and  stopper  T  just 
before  the  stream  of  breath  stops,  and  shake  for  one  minute  in  such 
a  manner  that  the  plasma  is  distributed  as  completely  as  possible 
about  the  walls.  After  the  shaking  has  lasted  a  minute,  blow  a 
fresh  portion  of  the  alveolar  air  through  the  beads  into  the  fun- 
nel and  shake  for  one  minute. 

The  C02  (Fig.  24)  apparatus  is  held  in  a  strong  clamp  W, 
which  is  lined  with  rubber,  and  the  lower  stop-cock  is  supported 
by  an  iron  rod,  which  is  also  covered  with  soft  rubber  tubing. 
The  apparatus  is  completely  filled  with  mercury.  Care  should  be 
taken  that  capillaries  A  and  F,  which  are  above  the  upper  stop- 
cock, are  also  filled  with  mercury.  There  should  be  no  air  bub- 
bles within  the  apparatus.  Six  dropping  bottles,  which  contain 
the  following  solutions,  should  be  at  hand  (see  Fig.  25)  : 

1.  Distilled  water. 

2.  Phenolphthalein   (1%  in  95%   alcohol). 

3.  Normal  ammonium  hydroxide. 

4.  Caprylic  alcohol. 

5.  Normal  sulphuric  acid. 

6.  Mercury. 


60 


BLOOD   AND    URINE    CHEMISTRY 


TESTS   FOR  ACIDOSIS   IN   BLOOD 


Gl 


Fig.  24. — CO2  apparatus. 


62 


BLOOD   AND   URINE    CHEMISTRY 


The  mercury  leveling  bulb  H  should  be  hung  by  wire  Z  on  ex- 
tension N,  about  on  the  level  with  the  lower  cock  J.  The  appa- 
ratus must  be  thoroughly  cleaned  before  the  determination  is 
started.  The  apparatus  can  be  tested  by  allowing  the  mercury 
to  run  down  and  then  forcing  it  up  by  raising  and  lowering  bulb 
//.  The  air  is  forced  out  and  the  mercury  is  caught  in  a  bottle 
as  shown  in  Fig.  26.  (This  is  done  until  there  is  not  a  single  air 
bubble  in  the  apparatus.)  Add  one  drop  of  phenolphthalein  to 
the  upper  cup  B  and  a  drop  or  two  of  the  ammonium  hydroxide. 
Now  dilute  this  with  about  i/2  c-c-  of  distilled  water  and  draw  off 
all  except  about  two  drops  of  the  alkaline  solution. 

Now  introduce  1  c.c.  of  the  saturated  plasma  into  the  cup  and 
allow  it  to  flow  under  the  alkaline  solution,  so  that  none  of  the 


Fig.  25. — Dropping  bottles  for  use  in  connection  with  CO2  determination. 

carbon  dioxide  escapes.  Turn  stop-cock  C  so  that  E  and  Z 
are  connected  and  allow  the  plasma  to  run  in  until  capillary  F  is 
exactly  filled.  Add  0.5  c.c.  of  distiUed  water  to  cup  B  and  then 
allow  to  run  down  to  capillary  F.  Eepeat  this,  taking  care  that 
no  air  enters  the  apparatus  with  the  liquid.  Now  admit  into  capil- 
lary F,  1  drop  of  caprylic  alcohol  to  prevent  foaming,  and  pour 
about  1.5  c.c.  of  the  sulphuric  acid  into  the  cup.  Admit  enough 
of  the  acid  into  the  apparatus,  carrying  the  caprylic  alcohol  along 
with  it,  so  that  the  total  volume  in  the  apparatus  is  exactly  to  the 
2.5  c.c.  mark.  Draw  off  the  excess  sulphuric  acid.  Now  place  a 
few  drops  of  mercury  in  cup  B  and  allow  to  flow  down  to  capillary 
F,  in  order  to  seal  same  and  make  it  capable  of  holding  an  absolute 
vacuum.  During  this  whole  operation,  the  lower  stop-cock  J 
should  remain  open,  and  when  the  apparatus  is  set  up,  it  should  be 


TESTS  FOR  ACIDOSIS  IN  BLOOD  63 

in  such  adjustment  that,  if  the  wire  /  which  is  connected  to  bulb  H 
is  lowered  to  hook  0,  the  mercury  will  run  to  the  mark  X  on  the 
figure  (Fig.  27),  care  being  taken  that  the  mercury  will  not  run 
into  fork  Y.  Place  wire  /-on  hook  0  and  allow  the  mercury  to  fall 
until  the  meniscus  of  the  mercury  has  dropped  to  the  50  c.c.  mark 
on  the  apparatus.  This  is  controlled  by  stop-cock  J.  The  bubbles 
of  C02  are  now  seen  escaping. 

In  order  to  completely  extract  the  carbon  dioxide,  remove  the 
apparatus  from  the  clamp  and  shake  by  turning  it  upside  down 
about  a  dozen  times.  (The  thumb  should  be  placed  over  cup  B  so 
as  not  to  lose  any  of  the  mercury.)  Then  replace  the  apparatus, 
the  mercury  leveling  bulb  H  still  being  at  the  low  level  0,  and  al- 
low the  solution  to  flow  into  the  small  bulb  below  the  lower  stop- 
cock (right  side) .  Drain  the  solution  out  of  the  portion  of  the  appa- 
ratus above  the  stop-cock  /  as  completely  as  possible,  but  without 
removing  any  of  the  gas  (the  last  drop  being  allowed  to  remain 
above).  Now  raise  the  mercury  bulb  H  in  the  left  hand,  and 
with  the  right  hand  immediately  turn  the  lower  stop-cock  J,  so 
that  the  mercury  is  admitted  to  the  upper  part  of  the  apparatus 
through  the  left-hand  entrance  of  the  stop-cock  without  readmit- 
ting the  watery  solution.  Hold  the  leveling  bulb  H  beside  the  ap- 
paratus so  that  its  mercury  level  corresponds  to  that  in  the  ap- 
paratus, and  the  gas  in  the  latter  is  under  atmospheric  pressure. 
A  few  hundredths  of  a  cubic  centimeter  of  water  will  float  on  the 
mercury  in  the  apparatus,  but  this  may  be  disregarded  in  leveling. 
The  calculation  of  the  result  into  terms  of  volume  percentage  of 
carbon  dioxide,  bound  as  carbonate  by  the  plasma,  is  quite  com- 
plicated and  we  consequently  use  the  direct  reading  from  the 
apparatus,  minus  .12. 

Plasma  of  normal  adults  yield  0.65  c.c.  to  .90  c.c.  of  gas  which 
is  the  direct  reading  on  the  apparatus.  If  .12  were  subtracted, 
the  normal  figures  would  be  53  to  78  in  terms  of  volume  per  cent 
of  carbon  dioxide  chemically  bound  by  the  plasma.  Figures  lower 
than  50  per  cent  in  adults  indicate  acidosis.  The  exact  calcula- 
tion of  the  result  into  terms  of  carbon  dioxide  bound  as  carbon- 
ate by  the  plasma  is  quite  complicated  and  consequently  the 
worker  is  advised  to  subtract  .12  from  his  reading  on  the  appa- 
ratus. The  result  thus  obtained  gives  approximately  (within  2  to 


64 


BLOOD   AND   URINE    CHEMISTRY 


Fig.   26. — CO2  apparatus  showing  air  being  forced 


TESTS    FOR    ACIDOSIS    IN   BLOOD 


Fig.  27.— CO2  apparatus.     Mercury  should  not  go  below  mark  X. 


66  BLOOD   AND   URINE    CHEMISTRY 

3  per  cent)  the  volume  per  cent  of  carbon  dioxide  bound  by  the 
plasma.  g 

Example. — Reading  on  the  Van  Slyke  apparatus  is  0.74  minus 
0.12  which  equals  0.62  per  cent  of  carbon  dioxide  bound  by  1  c.c. 
of  plasma,  For  100  c.c.  of  plasma  multiply  0.62%  by  100,  which 
equals  62%  (normal). 

Marriott,  Levy,  and  Rowntree  Method  for  the  Determination  of 
the  Hydrogen-ion  Concentration  of  the  Blood. 

Principle  of  the  Method. — Levy,  Rowntree,  and  Marriott1  state 
that  the  indicator  method  has  not  proved  of  great  value  in  the 
studies  of  hydrogen-ion  concentration  of  the  blood,  although  the 
reaction  of  inorganic  solutions  may  be  determined  accurately  by 
this  means.2  Different  indicators  show  their  color  changes  at  vary- 
ing degrees  of  hydrogen-ion  concentration:  for  example,  the  color 
of  methyl  orange  changes  from  pink  to  yellow  as  the  pH  of  its 
solution  changes  from  3  to  5.  At  intermediate  points,  various 
colors  may  be  obtained  and  a  certain  color  indicates  a  definite  pH. 
Similarly,  phenolphthalein  changes  from  colorless  to  pink  between 
pH8  and  pHIO  and  can  be  used  for  the  measurement  of  H-ion 
concentrations  between  these  two  points.  In  carrying  out  the  in- 
dicator method,  it  is  necessary  to  have  a  series  of  standard  solutions 
of  known  pH  and  an  indicator  exhibiting  easily  distinguishable 
color  changes  at  hydrogen-ion  concentrations  approximating  that 
of  the  solution  under  consideration.  It  is  then  simply  necessary 
to  add  equal  amounts  of  indicator  to  the  standard  solutions  and  to 
the  solution  being  tested  and  to  determine  which  of  the  colors  in 
the  standard  solutions  most  closely  matches  that  of  the  unknown 
solution. 

This  method  has  been  successfully  used  on  the  urine  by  Hender- 
son and  by  Walpole.  As  proteins  interfere  with  the  colors  of  many 
indicators,  and  as  both  blood  and  serum  possess  color,  it  has  been 
impossible  to  apply  the  method  directly  to  the  blood. 

It  seemed  probable  that  the  indicator  method  might  be  utilized 
for  blood,  provided  coloring  matters  and  proteins  could  be  ex- 
cluded by  means  of  dialysis.  If  blood  is  dropped  into  collodion  sacks 

JLevy,  Rowntree,  and  Marriott:     Arch,  of  Int.  Med.,  1915,  vol.  xvi,  p.  389. 
2S6renson:     Ergebn.   d.   Physiol.,  1912,   vol.   xii,   393.     A  full   description   of  indicators 
as  used  for  this  purpose. 


TESTS    FOR    ACIDOSIS    IN    BLOOD  67 

and  dialyzed  for  five  minutes,  the  dialysate  is  free  from  proteins 
and  coloring  matter,  but  contains  salts,  and  is  well  adapted  to 
the  use  of  indicators. 

Since  phenolsulphonphthalein  exhibits  definite  variations  in 
quality  of  color,  with  very  minute  differences  in  hydrogen-ion  con- 
centration between  pH6.4  and  8.4,  it  was  adopted  as  the  indicator 
in  this  method. 

Preparation  of  Standard  Colors. — Standard  phosphate  mixtures 
are  prepared  according  to  Sorenson  's  directions  as  follows : 

One-fifteenth  Molecular  Acid  or  Primary  Potassium  Phosphate. — 
Dissolve  9.078  grams  of  the  pure  recrystallized  salt  (KH,POJr 
in  freshly  distilled  water  and  make  up  to  one  liter. 

One-fifteenth  Molecular  Alkaline  or  Secondary  Sodium  Phos- 
phate.— Expose  the  pure  recrystallized  salt  (Na2HP04.12H20)  to 
the  air  for  from  ten  days  to  two  weeks,  protected  from  dust.  Ten 
molecules  of  water  of  crystallization  are  given  off  and  a  salt  of  the 
formula  Na2HP04.2H20  is  obtained;  dissolve  11.876  grams  of 
this  in  freshly  distilled  water  and  make  up  to  one  liter.  The 
solution  should  give  a  deep  rose  red  color  with  phenolphthalein. 
If  only  a  faint  pink  color  is  obtained,  the  salt  is  not  sufficiently 
pure. 

Mix  the  solutions  in  the  proportions  indicated  below  to  obtain 
the  desired  pH : 


PH  |  6.4|  6.6|  6.8|  7.0   7.1  j  7.2 j  7.3j  7.4J  7.5|  7.6J  7.7   7.8|  8.0J  8.2j  8.4 


Primary 

Potas. 

Phos.  c. 


,0: 


73.0  63.0  51.0  37.0  32.0  27.0  23.0  19.0  15.8  13.2  11.0   8.8 


5.6   3.2 


2.0 


Secondary 

Sodium 
Phos 


.  c. 


27.0  37.0  49.0  63.0  68.0  73.0  77.0  81.0  84.2  86.8  89.0  91.2  94.4  96.8  98.0 


Place  three  c.c.  of  each  of  the  solutions  in  suitable  small  test 
tubes  (100x10  mm.,  inside  measurement).  Add  five  drops  of  an 
aqueous  0.01  per  cent  solution  of  phenolsulphonphthalein  to  each 
tube.  Seal  off  the  tops.  The  series  of  colors,  representing  differ- 
ent concentrations  of  hydrogen-ions,  constitutes  the  standards  for 
comparison  of  color  in  carrying  out  the  determination. 

Preparation  of  Sacks. — Dissolve  one  ounce  of  collodion  (An- 
thony's negative  cotton)  in  500  c.c.  of  a  mixture  of  equal  quanti- 
ties of  ether  and  ethyl  alcohol.  The  solid  swells  up  and  dissolves 


68  BLOOD   AND   URINE    CHEMISTRY 

with  occasional  gentle  shakings,  in  forty-eight  hours.  As  a  small 
amount  of  brown  sediment  separates  out  at  first,  the  solution  should 
stand  for  at  least  three  or  four  days,  after  which  the  clear  super- 
natant solution  is  ready  for  use.  Fill  a  small  test  tube  (120  by  9 
mm.,  inside  measurement)  with  this  mixture,  invert,  and  pour  out 
half  the  contents.  The  tube  is  then  righted,  and  the  collodion  al- 
lowed to  fill  "the  lower  half  again.  Invert  a  second  time  and  rotate 
on  its  vertical  axis,  the  collodion  being  drained  off.  Care  must 
be  taken  to  rotate  the  tube,  in  order  to  secure  a  uniform  thickness 
throughout.  Clamp  the  tube  in  the  inverted  position  and  allow  to 
stand  for  ten  minutes,  until  the  odor  of  ether  finally  disappears. 
Fill  it  five  or  six  times  with  cold  water,  or  allow  it  to  soak  five 
minutes  in  cold  "water.  Run  a  knife  blade  around  the  upper  rim, 
so  as  to  loosen  the  sack  from  the  rim  of  the  test  tube,  and  run  a 
few  cubic  centimeters  of  water  down  between  the  sack  and  the 
glass  of  the  tube.  Extract  the  tu.be  by  gentle  pulling,  after  which 
preserve  by  complete  immersion  in  water. 

The  Salt  Solution  Used  in  the  Method. — Dialyze  the  blood  or 
serum  against  an  0.8  per  cent  sodium  chloride  solution. 

Before  applying  the  test,  it  is  necessary  to  ascertain  that  the 
solution  is  free  from  acids  other  than  carbonic.  To  determine  this, 
place  a  few  cubic  centimeters  of  the  salt  solution  in  a  Jena  test  tube 
and  add  one  or  two  drops  of  the  indicator,  whereupon  a  yellow 
color  will  appear.  On  boiling,  carbon  dioxide  is  expelled,  and  the 
solution  loses  its  lemon  color  and  takes  on  a  slightly  brownish 
tint.  In  the  absence  of  this  change,  other  acids  are  present,  and 
the  salt  solution  is  therefore  not  suitable.  If,  on  the  other  hand, 
on  adding  the  indicator,  pink  at  once  appears,  the  solution  is  alka- 
line and  hence  cannot  be  used. 

Technic  of  Method.— The  technic  can  be  carried  out  on  either 
serum,  plasma,  whole,  or  defibrinated  blood.  The  work  must  be 
done  in  a  room  free  from  fumes  of  acids  or  ammonia. 

Run  one  to  three  c.c.  of  clear  serum  or  of  blood,  by  means  of  a 
blunt  pointed  pipette,  into  a  dialyzing  sack  which  has  been  washed 
inside  and  outside  with  salt  solution  and  which  has  been  tested  for 
leaks  by  filling  with  the  salt  solution.  Lower  the  sack  into  a  small 
test  tube  (100  by  100  mm.,  inside  measurement)  containing  3  c.c. 
of  the  salt  solution,  until  the  fluid  on  the  outside  of  the  sack  is  as 
high  as  on  the  inside.  Allow  from  five  to  ten  minutes  for  dialysis. 


TESTS- FOR  ACIDOSIS  IN  BLOOD  69 

Remove  the  collodion  sack  and  add  5  drops  of  the  indicator  thor- 
oughly mixed  with  the  dialysate.  Then  compare  the  tube  with  the 
series  of  standards  until  the  corresponding  color  is  found,  which 
indicates  the  hydrogen-ion  concentration  present  in  the  dialysate. 

These  tests  have  been  carried  out  with  3  c.c.  of  blood  or  serum. 
The  same  results  are  obtained  with  1  c.c.  of  blood  or  serum  on 
the  inside  of  the  sack  and  with  this  amount  it  is  immaterial  whether 
there  is  1  or  3  c.c.  of  salt  solution  on  the  outside. 

Comparison  of  Tubes  With  Standards. — For  this,  a  good  light 
(natural  or  artificial)  and  a  white  background  are  requisites. 
Readings  must  be  made  immediately.  The  tube  matching  most 
closely  is  selected  and  also  the  tubes  on  either  side  of  it.  Those 
are  critically  inspected  against  a  white  background.  Changing  the 
order  of  the  tubes  often  makes  differences  more  apparent. 

Controls  of  the  Method. — Repeated  duplicate  determinations  on 
the  same  samples  of  blood  and  of  serum  have  convinced  Marriott 
and  his  co-workers  that  the  limits  of  error  are  very  slight:  for  ex- 
ample, the  serum  from  a  case  of  mild  acidosis  (using  quantities 
of  serum  varying  from  1  to  3  c.c.  and  dialyzing  for  from  five  to 
fifteen  minutes)  gave  the  following  series  of  readings:  7.55,  7.55, 
7.55,  7.55,  7.6,  7.55,  7.55,  7.55,  7.55,  7.55.  The  oxalated  whole  blood 
from  the  same  case  gave  the  following  readings  under  similar  con- 
ditions: 7.25,  7.25,  7.25,  7.25,  7.2,  7.25,  7.25,  7.3,  7.25,  7.25.  7.25,- 
7.25,  7.25,  7.25. 

In  order  to  test  out  the  effect  of  the  variations  in  the  sacks  used, 
a  number  of  determinations  were  made  on  the  same  sample  of 
serum  with  the  following  results :  ordinary  thin  sack,  7.7 ;  thick 
sack,  7.7 ;  opaque,  irregular  sack,  7.7 ;  ordinary  thin  sack,  7.65 ;  a 
very  thick  sack,  7.7.  A  series  of  six  normal  serums  were  run 
through,  3  c.c.  and  1  c.c.  portions  being  used  for  dialysis.  In  every 
instance  identical  readings  were  obtained. 

A  brief  word  of  explanation  may  be  given  for  those  unaccus- 
tomed to  the  physicochemical  methods  of  expressing  the  reaction 
of  a  solution.  A  solution  is  acid  when  it  contains  an  excess  of 
hydrogen  over  hydroxyl-ions,  neutral  when  hydrogen-  and  hy- 
droxyl-ions  are  in  equal  numbers,  and  alkaline  when  hydroxyl-ions 
predominate.  An  acid  of  "normal"  strength  contains,  in  one  liter, 
a  gram  of  hydrogen  capable  of  forming  hydrogen-ions  and  its 
strength  may  be  expressed  as  1  N.  Diluting  such  a  solution  ten 


70  BLOOD   AND   URINE    CHEMISTRY 

times,  we  would  have  1/10  N  or  a  solution  containing  1/10  gram 
of  actual  or  potential  hydrogen-ions  to  the  liter.  Continuing  the 
process  of  dilution  until  1/10,000,000  normal  acid  is  obtained,  we 
would  have  in  such  a  solution  1/10,000,000  gram  of  hydrogen-ions. 
Pure  water,  however,  dissociates  to  form  hydrogen-  and  hydroxyl- 
ions,  and  at  20°  C.  contains  approximately  1/10,000,000  gram  of 
hydrogen-ions  to  the  liter  and  an  equivalent  amount  of  hydroxyl- 
ions  (that  is,  17  gm.).  That  is  to  say,  pure  water,  our  standard 
-of  neutrality,  is  1/10,000,000  N  acid  and  also  1/10,000,000  N  alka- 
line. To  avoid  writing  large  figures  it  is  customary  to  use  the 
logarithmic  notation  and  to  express  1/10,000,000  N  as  10— 7N  or 
more  conveniently,  as  suggested  by  Sorenson,  to  drop  the  10  and 
minus  sign  and  say  pH7.  If  we  have  less  than  1/10,000,000 
gram  of  hydrogen-ions  in  one  liter  the  solution  is  less  acid  than 
water,  that  is,  it  is  alkaline — so,  pH8  means  actually  1/10,000,000 
N  alkali.  The  higher  the  exponent,  the  more  the  alkaline,  or  what 
is  saying  the  same  thing,  the  less  acid  the  solution. 
To  sum  up: 

pHl=N/10  acid. 


pH6=N/l,000,000  acid. 
pH7r=NEUTRALITY. 
pH8=N/l,000,000  alkali. 


pH14=rN/10  alkali. 

The  reaction  of  the  blood  serum  varies  approximately  between 
pH7  and  pH8,  the  neutral  point,  pH7  being  reached  only  in  severe 
uncompensated  acidosis,  and  a  reaction  of  pH8  being  attained 
perhaps  only  after  administration  of  alkalies. 

The  Determination  of  the  Alkali  Reserve  of  the  Blood  Plasma. 

Marriott  has  recently3  published  a  method  which  gives  the  hydro- 
gen-ion concentration  of  the  dialysate  of  blood  serum  after  re- 
moval of  the  carbon  dioxide,  that  is  in  a  measure  a  modification  of 
the  indicator  analysis  of  the  preceding  test,  but  is  more  accurate 
and  gives  more  information  than  that  method.  This  method  serves 


'Marriott:     Arch.  Int.  Med.,  June,  1916,  vol.  xvii,  pp.  840-851. 


TESTS  FOR  ACIDOSIS  IN  BLOOD  71 

for  the  detection  and  accurate  quantitative  estimation  of  the  de- 
gree of  the  acidosis. 

Apparatus  Required.— Set  of  tubes  containing  standard  phos- 
phate mixtures;  a  solution  of  phenolsulphonphthalein  in  0.8  per 
cent.  Sodium  chloride ;  collodion  sacks ;  pipette  to  measure  0.5  c.c. ; 
small  test  tubes  for  dialyzing  and  aerating;  atomizer  bulb;  glass 
tube  or  pipette  drawn  out  to  a  fine  capillary  point;  color  com- 
parison box. 

Preparation  of  Phosphate  Mixtures — One-fifteenth  Molecular 
Acid  Potassium  Phosphate. — Dissolve  9.078  gms.  of  the  pure  re- 
crystallized  salt  (KH2POJ  in  freshly  distilled  water.  Add  200  c.c. 
of  0.01  per  cent  phenolsulphonphthalein  and  make  up  the  whole  to 
1  liter  with  distilled  water. 

One-fifteenth  Molecular  Alkaline  Sodium  Phosphate. — Expose 
the  pure,  recrystallized  salt  (Na2HPO4.12H20)  to  the  air  for  from 
ten  days  to  two  weeks,  protected  from  dust.  Ten  molecules  of 
water  of  crystallization  are  given  off  and  a  salt  of  the  formula 
Na2HP04.2H20  is  obtained.  Dissolve  11.876  gms.  of  this  salt 
in  distilled  water.  Add  200  c.c.  of  0.01  per  cent  of  phenolsulphon- 
phthalein and  make  up  the  whole  to  one  liter.  The  exact  amount 
of  indicator  is  immaterial,  provided  the  'same  amount  of  indicator 
is  added  to  each  of  fhe  phosphate  solutions,  and  a  corresponding 
amount  is  added  to  the  salt  solution,  to  be  subsequently  described. 
Add  a  small  crystal  of  thymol  to  each  solution  to  prevent  the 
growth  of  molds.  The  solutions  should  be  preserved  in  Jena  or 
Non-sol  glass  vessels.  Mix  the  solutions  in  the  proportions  indi- 
cated below  to  obtain  the  desired  pH. 

pH  |    7.0  |    7.2  |    7.4  |    7.6  |    7.8  |    8.0  |    8.2  |    8.4  |    8.6 

Primary  sod,  phos.,  c.c.  |  37.0  |  27.0  |  19.0  |  13.2  |  8.8  |  5.6  |  3.2  |  2.0  |^0 
Secondary  sod,  phos^c.c.  |  63.0  |  73.0  |  81.0j^678  |  91.2  |  94.4  |  96.8  |  98.0  |  99.0 

Place  these  solutions  in  small  test  tubes,  approximately  100  mm. 
long  by  8  mm.,  internal  diameter,  of  glass  that  does  not  readily 
give  off  alkali.  The  tubes  are  stoppered  or  sealed  off.  They 
should  be  kept  in  a  dark  place  when  not  in  use.  Under  these  con- 
ditions, the  solutions  retain  their  colors  for  long  periods  of  time. 

Preparation  of  Salt  Solution.— Dissolve  8  gms.  of  chemically 


72  BLOOD   AND   URINE    CHEMISTRY 

pure  sodium  chloride  in  distilled  water.  Add  220  c.c.4  of  0.01  per 
cent  phenolsulphonphthalein  solution  and  make  up  the  whole  to  one 
liter  with  distilled  water.  The  solution  should  contain  no  free 
alkali  and  no  acid  other  than  carbonic.  Test  the  solution  by  boil- 
ing a  little  of  it  for  a  minute  or  so  in  a  Jena  glass  test  tube,  in 
order  to  expel  carbonic  acid.5  Cool  the  solution  quickly  under  the 
tap  and  compare  with  the  phosphate  standards.  Its  reaction 
should  be  7.0.  If  the  reaction  differs  from  this,  it  may  be  cor- 
rected by  the  addition  of  a  few  drops  of  very  dilute  acid  or  alkali 
to  the  whole  solution.  The  salt  solution  must  be  kept  in  a  vessel 
of  Jena  or  Non-sol  glass,  or  in  a  vessel  of  ordinary  glass  that  has 
been  well  paraffined  on  the  inside. 

METHOD  OP  DETERMINATION. — The  determination  must  be  car- 
ried out  in  a  room  free  from  acid  or  ammonia  fumes.  Either 
serum,  oxalated  plasma,  or  blood  may  be  used.  Serum  is  to  be 
preferred,  as  the  addition  of  oxalate,  unless  exactly  neutral,  intro- 
duces a  source  of  error.  The  blood  should  be  collected  in  a  small 
tube  and  the  serum  separated  as  quickly  as  possible,  preferably 
by  centrifuging.6  Hemolysis  must  be  avoided. 

Pipette  exactly  0.5  c.c.  of  serum  into  one  of  the  small  collodion 
sacks,  which  has  previously  been  washed  inside  and  out  with  the 
salt  solution.7  Lower  the  sack  into  a  small  test  tube,  approximately 
8  mm.  internal  diameter  and  50  mm.  long,  containing  2  c.c.  of  the 
indicator  salt  solution.  The  level  of  the  fluid  on  the  outside  of  the 
sack  should  be  at  least  as  high  as  that  on  the  inside.  At  the  end 
of  seven  minutes  remove  the  sack  and  transfer  the  dialysate  to  a 
clean  test  tube  100  to  140  mm.  long  and  having  the  same  diameter 
as  the  tubes  containing  the  phosphate  standards.  A  rapid  current 
of  air  is  bubbled  through  the  solution  in  order  to  remove  carbon 
dioxide.  This  is  accomplished  by  means  of  an  atomizer  bulb  con- 

"The  concentration  of  indicator  in  the  salt  solution  is  purposely  made  10%  greater 
than  in  the  phosphate  mixtures,  as  Buring  the  dialysis  a  certain  amount  of  indicator  is 
lost  by  passing  into  the  sack. 

5If  boiled  in  a  soft  glass  tube,  alkali  is  given  off  from  the  glass  and  the  solution  is 
colored  pink.  Instead  of  boiling  to  remove  carbon  dioxide,  the  solution  may  be  aerated 
with  a  current  of  air  that  has  been  freed  from  carbon  dioxide  by  passing  through  a 
strong  solution  of  sodium  hydroxide. 

6If  carbon  dioxide  escapes  from  the  -plasma  as  a  result  of  shaking  or  allowing  the 
blood  to  remain  exposed  to  the  air,  a  passage  of  alkali  from  the  plasma  into  the  cells 
occurs  with  a  resultant  slight  diminution  in  the  alkali  reserve  of  the  plasma.  Once  the 
plasma  or  serum  is  separated  from  the  corpuscles,  loss  of  carbon  dioxide  is  without  effect 
on  the  alkali  reserve. 

7In  washing  the  sack,  no  part  but  the  top  edge  should  be  touched  with  the  fingers  The 
sack  is  emptied  by  tipping  it  with  a  clean,  glass  rod  or  with  a  microscopic  slide.  Sacks 
may  be  used  more  than  once,  providing  they  are  thoroughly  washed  with  salt  solution 
after  each  test. 


TESTS  FOR  ACIDOSIS  IN  BLOOD  73 

nected  with  a  narrow  glass  tube  drawn  out  to  a  capillary  point. 
The  air  current  should  be  as  rapid  as  possible  without  blowing 
liquid  out  of  the  test  tube.8  Continue  blowing  for  three  minutes 
and  then  compare  the  color  in  the  tube  with  that  in  the  standard 
phosphate  tubes,  interpolating  when  necessary.  The  reading  is  a 
measure  of  the  reserve  alkalinity.  For  convenience  of  expression 
this  value  is  referred  to  as  the  "RpH"  of  the  serum,  to  differen- 
tiate it  from  the  "pH"  as  determined  in  the  method  previously 
described  by  Levy,  Rowntree,  and  Marriott. 

RESULTS  OBTAINED. — Normal  Individuals.  The  serums  of  a 
large  number  of  normal  adults  were  examined  by  the  method  de- 
scribed. In  every  instance  the  RpH  was  found  to  be  8.5  db  0.05, 
provided  the  subjects  examined  were  on  a  general  mixed  diet.  A 
normal  adult's  serum  drawn  after  a  fast  of  sixteen  hours  gave  a 
reading  of  8.35.  The  serums  of  infants  gave  values  slightly  lower 
than  those  of  adults.  For  normal  infants  under  one  year  of  age,  a 
value  of  8.3  for  the  RpH  of  the  serum  was  not  infrequently  en- 
countered. This  may  be  due  partly  to  the  fact  that  infant 's  blood 
is  usually  obtained  by  cupping;  the  lower  value,  however,  is  more 
likely  an  evidence  of  the  tendency  towards  acidosis  that  is  known 
to  be  present  in  infants. 

This  accords  well  with  the  observed  fact  that  the  carbon  dioxide 
tension  in  the  alveolar  air  of  infants  is  lower  than  that  of  adults, 
and  that  the  combined  carbon  dioxide  of  the  plasma  is  less  in  in- 
fants and  that  the  ammonia  co-efficient  in  the  urine  is  often  higher. 
This  slight  acidosis  might  well  be  the  result  of  the  more  active 
metabolism  of  infants,  leading  to  a  proportionately  greater  pro- 
duction of  acids. 

ACIDOSIS. — A  series  of  cases  exhibiting  clinical  or  laboratory  evi- 
dences of  acidosis  were  studied.  The  cases  included  nephritis  and 
diabetes  in  adults,  and  nephritis,  recurrent  and  idiopathic  aceto- 
nemia,  and  severe  diarrheas  in  children.  The  diarrhea!  cases  were 
of  the  type  described  by  Howland  and  Marriott. 

In  all  the  cases  of  acidosis  studied,  the  RpH  of  the  serum  showed 
deviations  from  the  normal.  The  more  severe  the  acidosis,  as 
indicated  clinically  or  by  various  laboratory  methods,  the  lower 
were  the  figures  obtained  for  the  RpH.  Especially  striking  was  the 


8Foaming  rarely  occurs.  It  may  be  present  as  a  result  of  allowing  some  serum  to  spill 
over  the  outside  of  the  sack.  In  case  foaming  is  great  enough  to  be  troublesome,  it  may  be 
prevented  by  adding  a  drop  of  octyl  alcohol  or  toluol. 


74  BLOOD   AND   URINE    CHEMISTRY 

parallelism  between  alveolar  carbon  dioxide  tension  and  the  RpH. 
The  two  values  should  correspond,  as  explained  above,  provided  the 
respiratory  center  does  not  vary  in  its  excitability  and  the  pul- 
monary epithelium  is  not  damaged  in  such  a  way  as  to  prevent 
equilibrium  being  attained  between  the  air  in  the  pulmonary  alveoli 
and  the  blood  in  the  pulmonary  capillaries.  Thus  a  hyperexcitable 
respiratory  center  should  lead  to  a  low  alveolar  carbon  dioxide 
tension,  with  a  coincident  normal  alkali  reserve.  A  diminished 
permeability  of  the  pulmonary  epithelium  would  result  in  a  lower- 
ing of  carbon  dioxide  tension  in  the  alveolar  air,  but  not  necessarily 
to  a  diminution  in  the  alkali  reserve  of  the  plasma. 

In  a  number  of  instances  the  combined  carbon  dioxide  of  the 
plasma  was  determined  according  to  the  method  described  by  Van 
Slyke.  The  results  obtained  were  in  a  general  way  proportional 
to  the  RpH  of  the  serum.  The  RpH  invariably  showed  an  increase 
following  administration  of  alkalies,  but  did  not  necessarily  reach 
its  normal  value.  It  was  in  connection  with  the  alkali  therapy 
that  Marriott  found  the  method  of  especial  value,  as  it  gave  infor- 
mation as  to  the  probable  amount  of  alkali  needed  to  replenish  the 
reserve.  A  determination  following  the  administration  of  alkali 
showed  whether  the  amount  was  sufficient. 

Interpretation  of  Results.— The  values  obtained  for  the  RpH 
of  the  serum  may,  in  the  light  of  his  experience,  be  interpreted  as 
follows : 

Values  for  the  RpH  of  from  8.4  to  8.55  correspond  to  alveolar 
carbon  dioxide  tensions  of  from  38  to  45  mm.,  and  are  to  be  con- 
sidered as  normal  values. for  adults.  Values  between  8.0  and  8.3 
correspond  to  alveolar  carbon  dioxide  tensions  of  from  28  to  35 
mm.,  and  indicate  a  moderate  degree  of  acidosis. 

When  the  value  for  RpH  is  as  low  as  7.7,  corresponding  to  an 
alveolar  carbon  dioxide  tension  of  20  mm.,  the  individual  is  in  im- 
minent danger.  During  coma,  an  RpH  as  low  as  7.3  corresponding 
to  an  alveolar  air  of  11  mm.,  was  observed.  In  infants  under  one 
year  of  age  a  value  for  RpH  of  8.3,  corresponding  to  35  mm.  ten- 
sion in  the  alveolar  air,  is  not  to  be  considered  abnormal. 

It  has  been  Marriott's  experience  in  general  that  unless  the  RpH 
of  the  serum  is  below  7.9,  the  acidosis  may  be  successfully  combated 


TESTS   FOR  ACIDOSIS   IN   BLOOD  75 

by  dietetic  regulation  or  by  the  administration  of  alkali  by  mouth. 
When  the  RpH  of  the  serum  falls  below  7.9,  intravenous  adminis- 
tration of  alkali  is  usually  indicated. 

BIBLIOGRAPHY. 

Beddard,  Pembery,  and  Spriggs:     Jour.  Physiol.,  1908,  p.  37. 

Boothby  and  Peabody:     Arch.  Int.  Med.,  1914,  p.  497. 

Higgins:     Carnegie  Inst.  of  Washington,  1915,  pub.  203,  p.  168. 

Higgins  and  Means:     Jour.  Pharm.  and  Exper.  Therap.,  1915,  vol.  vii,  p.  1. 

Higgins,  Peabody,  and  Fitz:     Jour.  Med.  Research,  1916,  vol.  xxiv,  p.  263. 

Howland  and  Marriott:     Bull.  Johns  Hopkins  Hosp.,  1916,  vol.  xxvii,  p.  63. 

Howland  and  Marriott:     Am.  Jour.  Dis.  Child.,  May,  1916. 

Levy  and  Rowntree :     Arch.  Int.  Med.,  1916,  vol.  xvii,  p.  525. 

Levy,  Rowntree  and  Marriott:     Arch.  Int.  Med.,  1915,  vol.  xvi,  p.  389. 

Marriott:     Arch.  Int.  Med.,  1916,  vol.  xvii,  p.  840;   Jour.  Am.  Med.  Assn., 

1916,  vol.  Ixvi,  p.  1594. 

McClendon :     Jour.  Biol.  Chem.,  1916,  vol.  xxiv,  p.  519. 
McClendon  and  Magoon :     Jour.  Biol.  Chem.,  vol.  xxv,  p.  669. 
Peabody :     Am.  Jour.  Med.  Sc.,  1916,  vol.  cli,  p.  184. 
Plesch:     Ztschr.  f.  exper.  Path.  u.  Therap.,  1909,  vol.  vi,  p.  380. 
Stillman :     Am.  Jour.  Med.  Sc.,  1916,  vol.  cli,  p.  507. 
Van  Slyke:     Unpublished  data. 


PART  II. 
CHEMICAL  ANALYSIS  OF  URINE 


CHAPTER  XIII. 
TOTAL  NITROGEN. 

The  method  given  below  is  a  slight  modification  of  the  method 
given  by  Myers  and  Fine  which  in  turn  is  a  modification  of  the 
colorimetric  method  of  Folin  and  Farmer.  The  only  difference  in 
technic  is  that  of  adding  peroxide  of  hydrogen  to  hasten  oxida- 
tion, which  considerably  shortens  the  time  of  making  the  test. 
In  the  method  of  Myers  and  Fine,  fully  fifteen  to  twenty  minutes 
is  required  to  complete  the  determination.  As  here  described,  the 
estimation  may  be  completed  in  from  five  to  ten  minutes.1 

For  the  determination,  an  amount  of  urine  sufficient  to  con- 
tain between  0.35  and  0.75  mgms.  nitrogen  is  required.  This  is 
usually  obtained  by  a  1  to  25  dilution  of  urine,  although  some- 
times a  dilution  of  1  to  10  is  sufficient,  as  indicated  by  a  low  spe- 
cific gravity.  Take  1  c.c.  of  urine  with  an  Ostwald-Folin  pipette 
and  dilute  to  25  c.c.  with  distilled  water  in  a  volumetric  flask. 
After  mixing  thoroughly,  place  1  c.e.  of  this  material  in  a  thin 
glass  test  .tube,  to  which  is  added  5  to  7  drops  (0.1  c.c.)  of  con- 
centrated sulphuric  acid,  50  to  100  mgms.  of  potassium  sulphate, 
and  a  drop  of  copper  sulphate  (10%).  Now  boil  the  tube  by  hand 
(or  in  the  apparatus  as  shown  in  Fig.  17)  with  continued  shaking 
(if  boiled  in  apparatus  no  shaking  is  required)  until  the  con- 
tents become  dark  brown,  and  then,  while  the  tube  is  warm  (not 
hot),  add  a  drop  of  hydrogen  peroxide,  and  if  not  clear,  heat 
about  one  minute  until  clear.  It  is  this  part  of  the  technic  that 
we  have  modified ;  namely,  the  addition  of  peroxide  of  hydrogen. 
"When  digestion  is  completed,  allow  the  tube  to  cool  for  one  min- 
ute and  then  wash  into  a  50  c.c.  volumetric  flask  or  accurate  50 

Kiradwohl  and  Blaivas:     Jour.  Am.  Med.  Assn.,  Sept.  9,  1916,  vol.  Ixvii,  p.  809. 


TOTAL    NITROGEN  77 

c.c.  graduate  (A)  with  about  35  c.c.  distilled  water.  Pipette  5  c.c. 
of  ammonium  sulphate  solution2  containing  1  mgm.  of  nitrogen 
per  5  c.c.  with  an  Ostwald-Folin  pipette  into  a  50  c.c.  volumetric 
flask  (B),  if  the  Hellige  colorimeter  is  to  be  employed,  and  add 
about  30  c.c.  of  distilled  water.  Dilute  10  c.c.  of  the  modified 
Nessler's  solution3  with  40  c.c.  of  distilled  water  just  previous 
to  use,  mix,  and  make  up  at  once  the  material  in  the  second  volu- 
metric flask  (standard)  to  volume  with  the  diluted  Nessler's  so- 
lution. The  flask  (A),  unknown,  is  then  made  up  to  volume  with 
the  diluted  Nessler's  solution,  as  in  flask  B,  except  that  the  Ness- 
ler's solution  is  added  slowly  at  first  while  rotating  the  flask,  un- 
til the  alkali  of  the  Nessler's  solution  has  neutralized  the  sul- 
phuric acid.  Fill  a  dry,  glass-stoppered  wedge  for  the  Hellige 
colorimeter  with  the  standard  solution  (see  Plate  I  for  the  stand- 
ard color  of  1  mgm.  of  nitrogen)  and  adjust  in  the  colorimeter. 
Next  place  slightly  over  2  c.c.  of  the  unknown  solution  in  the 
empty  cup,  insert  in  the  colorimeter,  and  match  the  colors,  prefer- 
ably with  a  north  light.  The  amount  of  nitrogen  in  1/25  c.c.  of 
urine  may  be  ascertained  in  the  following  table  from  which  the 
nitrogen  content  of  the  specimen  of  urine  under  examination  may 
be  easily  computed. 

Since  the  figures  in  the  table  are  given  for  a  dilution  of  100  c.c., 
and  the  dilution  here  employed  is  50  c.c.,  the  result  obtained 
should  be  divided  by  2. 

Example. — The  twenty-four  hour  specimen  of  urine  contains 
1500  c.c.  Our  dilution  is  1  to  25.  Suppose  the  dilution  is  50. 
Reading  is  75,  which  is  equivalent  to  0.56  mgm.  per  dilution  of 
100  c.c.  Divide  by  2  equals  0.28  (our  dilution  is  50)  ;  multiply 
0.28  by  25  to  obtain  the  amount  in  1  c.c.  which  is  7  mgms.,  multi- 
plied by  1500  is  10,500  mgms.  or  10.5  grams  of  nitrogen  in  1500 
c.c.  urine. 


2This  standard  solution  is  prepared  by  dissolving  0.944  gm.  of  ammonium  sulphate  in 
distilled  water  and  making  up  to  100  c.c. 

3For  one  liter  we  need  100  gms.  of  mercuric  iodide,  50  gms.  of  potassium  'iodide,  and 
200  gms.  of  potassium  hydroxide.  Place  the  mercuric  iodide  and  potassium  iodide,  both 
finely  powdered,  into  a  liter  volumetric  flask  and  add  about  400  c.c.  of  distilled  water. 
Now  dissolve  the  potassium  hydroxide  in  500  c.c.  distilled  water,  cool  thoroughly,  and 
add  with  constant  shaking  to  the  mixture  in  the  flask.  Then  make  up  to  one  liter  with 
water.  This  usually  becomes  perfectly  clear.  Keep  at  37°  C.  in  incubator  over  night  or 
until  the  yellowish  white  precipitate  which  may  settle  out  is  thoroughly  dissolved  and 
only  a  small  amount  of  the  dark  brownish  precipitate  remains.  The  solution  is  now 
ready  to  be  siphoned  off  and  used. 


78 


BLOOD   AND   URINE    CHEMISTRY 


TABLE  VI4 


ESTIMATION  OF  NITROGEN  WITH  THE  HELLIGE  COLORIMETER 


COLORI- 

NITROGEN 

COLORI- 

NITROGEN 

COLORI- 

NITROGEN 

METRIC 

MGMS.  PER 

METRIC 

MGMS.  PER 

METRIC 

MGMS.  PER 

READING 

DILUTION 

BEADING 

DILUTION 

READING 

DILUTION 

OF  100  C.C. 

OF  100  C.C. 

OF  100  C.C. 

20 

1.73 

.   40 

1.31 

60 

0.89 

21 

1.71 

41 

1.29 

61 

0.87 

22 

1.69 

42 

1.27 

62 

0.85 

23 

1.67 

43 

1.25 

63 

0.83 

24 

1.65 

44 

1.23 

64 

0.81 

25 

1.62 

45 

1.20 

65 

0.78 

26 

1.60 

46 

1.18 

.  66 

0.76 

27 

1.58 

47 

1.16 

67 

0.74 

28 

1.56 

48 

.14 

68 

0.72 

29 

1.54 

49 

.12 

69 

0.70 

30 

1.52 

50 

.10 

70 

0.67 

31 

1.50 

51 

.08 

71 

0.65 

32 

1.48 

52 

1.06 

72 

0.63 

33 

1.46 

53 

1.04 

73 

0.61 

34 

1.44 

54 

1.02 

74 

0.59 

35 

1.41 

55 

0.99 

75 

0.56 

36 

1.39 

56 

0.97 

76 

0.54 

37 

1.37 

57 

0.95 

77 

0.52 

38 

1.35 

58 

0.93 

78 

0.50 

39 

1.33 

59 

0.91 

79 

0.48 

4Myers   and   Fine:      Chemical   Composition   of   the    Blood 
York,   1915. 


Health   and   Disease,    New 


CHAPTER  XIV. 
UREA. 

Dilute  the  urine  1  to  10  with  distilled  water.  Pipette  2  c.c.  of 
the  diluted  urine  into  a  test  tube  of  such  dimensions  that  it  will 
easily  slip  into  a  100  c.c.  graduated  cylinder  (no  lip),  add  about 
0.1  gm.  of  urease  and  incubate  the  contents  in  a  beaker  of  water 
at  50°  C.  for  one-half  hour.  At  the  end  of  this  time,  add  two 
drops  of  caprylic  alcohol  or  1  c.  c.  of  amylic  alcohol  to  prevent 
foaming  in  aeration. 

We  now  call  attention  to  the  manner  of  setting  up  the  glass- 
ware for  the  continuation  of  this  test  (see  Fig.  15).  The  chemistry 
of  this  estimation  is  about  as  follows :  the  enzyme  urease  converts 
urea  into  ammonium  carbonate.  The  ammonia  is  then  liberated 
by  aeration  in  the  presence  of  sodium  carbonate  in  excess  and  goes 
over  into  the  hydrochloric  acid  as  ammonium  chloride.  This 
can  be  determined  colorimetrically  by  the  use  of  Nessler's  re- 
agent. There  should  be  two  cylinders  for  each  sample  of  urine. 
If  more  than  one  urine  is  to  be  examined,  these  cylinders  may  be 
run  in  series,  two  for  each  test.  One  cylinder  is  graduated,  the 
other  nongraduated.  A  two-hole  rubber  stopper  is  placed  in  each 
cylinder.  Cylinder  1  (A- A')  is  graduated  and  is  connected  with 
the  suction.  Cylinder  2  (B-B')  is  nongraduated  and  is  connected 
with  the  acid  wash  bottle  (C).  If  more  than  one  urine  is 
under  examination,  cylinder  2  is  connected  with  the  short  con- 
nection of  the  other  graduated  cylinder,  etc.  This  acid  wash 
bottle  is  simply  a  bottle  containing  sulphuric  acid  (10%)  placed 
at  the  end  of  the  outfit  to  prevent  ammonia  from  the  air  from 
gaining  entrance  into  the  test.  Cylinder  1  (A-A')  has  a  short 
tube  bent  at  right-angles  connected  to  the  suction  and  only  ex- 
tending in  the  cylinder  to  a  point  just  within  the  cylinder.  This  is 
tube  F-F'.  Tube  G-G'  extends  almost  to  the  bottom  of  cylinder  1. 
The  end  of  tube  G-G'  is  sealed  and  a  number  of  small  holes  are 
punched  in  its  side  with  platinum  wire  which  is  at  white  heat, 
provided  the  glass  is  only  moderately  hot.  Cylinder  2  has  a  right- 


80  BLOOD   AND   URINE    CHEMISTRY 

angle  tube  extending  to  a  point  just  below  the  stopper  (D) .  It  has 
another  tube  with  a  straight  open  end  dipping  into  the  test  tube 
(E)  and  running  out  to  be  connected  either  with  the  acid  wash 
bottle  extension  or  with  another  series  of  cylinders  in  case  more 
than  one  urine  is  under  examination.  Into  the  100  c.c.  graduated 
cylinder  (cylinder  1)  add  20  c.c.  distilled  water  and  2  to  3  drops 
of  10%  hydrochloric  acid.  This  is  now  closed  and  cylinder  2 
opened.  To  the  test  tube  containing  the  digested  urine  allow  an 
equal  volume  of  saturated  sodium  carbonate  to  slowly  run  down 
the  side  of  the  tube  under  the  urine.  Now  immediately  and  care- 
fully insert  the  tube  into  cylinder  2  and  immediately  close,  and 
then  carefully  and  tightly  seal  the  connection.  Start  the  suc- 
tion slowly  by  means  of  the  Chapman  pump  and  continue  slowly 
for  about  five  minutes,  and  then  increase  the  speed  of  the  suc- 
tion as  much  as  the  apparatus  will  stand.  Keep  up  the  aeration 
for  thirty  to  forty-five  minutes.  At  the  end  of  this  time  dis- 
connect the  cylinders,  and  cylinder  1  is  used  for  the  final  determi- 
nation. Remove  the  rubber  stopper  from  cylinder  1  and  wash 
down  the  tube  with  distilled  water  (2  to  3  c.c.). 

We  now  come  to  the  development  of  color.  Into  a  50  c.c. 
volumetric  flask,  pipette  5  c.c.  of  ammonium  sulphate  solution1 
containing  1  mgm.  of  nitrogen,  add  25  c.c.  distilled  water  and 
20  c.c.  Nessler's  solution2  diluted  1  to  5  (see  Plate  I  for  standard 
color  of  1  mgm.  of  nitrogen).  To  cylinder  1  containing  the  un- 
known in  the  form  of  ammonium  chloride,  add  from  10  to  25  c.c. 
of  diluted  Nessler's  solution  (1  to  5),  depending  upon  the  depth 
of  color,  and  dilute  to  50  c.c.,  100  c.c.,  etc.,  depending  upon  the 
color.  Make  the  colorimetric  reading  at  once  and  compare  and 
compute  from  the  table  for  the  estimation  of  nitrogen  with  the 
Hellige  colorimeter  (see  page  78). 

The  result  will  be  for  0.2  c.c.  of  urine  (urine  diluted  1  to  10  for 
this  test  and  2  c.c.  of  diluted  urine  taken  for  the  determination 
which  is  equivalent  to  0.2  c.c.  urine). 

Example. — The  twenty-four  hour  specimens  contain  1500  c.c. ; 
dilution  is  100 ;  reading  is  58.  Equivalent  from  table  is  0.93  mgms. 
in  0.2  c.c.  urine.  Multiply  by  5  equals  4.65  mgms.  in  1  c.c.  urine ; 

'See  footnote  2,  page  77. 
2See  footnote  3,  page  77. 


UREA  81 

multiply  by  1500  equals  6975  mgms.  in  1500  c.c.  urine  or  6.975 
grams  Urea  N  in  1500  c.c.  urine. 

The  amount  of  urea  is  computed  by  multiplying  the  urea  ni- 
trogen by  the  factor  2.14. 

Example. — Urea  nitrogen  from  above  equals  6.975  grams,  mul- 
tiplied by  2.14,  equals  14.9265  grams  of  urea  in  1500  c.c.  urine. 

To  obtain  an  accurate  figure  for  the  urea  nitrogen  it  is  necessary 
to  make  a  correction  for  the  amount  of  ammonia  nitrogen  origi- 
nally present. 


CHAPTER  XV. 
AMMONIA. 

An  amount  of  urine  sufficient  to  give  0.75  to  1.50  mgms.  of  am- 
monia nitrogen  should  be  employed.  With  normal  urines  2  c.c. 
will  generally  yield  the  desired  amount.  With  very  diluted  urines 
5  c.c.  may  be  required,  while  with  diabetic  urines,  rich  in 
ammonium  salts,  1  c.c.  may  be  excessive,  thus  requiring  dilution. 
Pipette  the  desired  amount  into  a  test  tube  about  200  mm.  in 
length  and  of  sufficient  diameter  so  that  it  will  slip  easily  into  a 
100  c.c.  graduated  cylinder  (no  lip). 

Aeration  is  carried  out  in  the  following  manner:  To  cylinder 
1  add  20  c.c.  of  distilled  water  and  2  to  3  drops  of  10%  hydro- 
chloric acid;  then  close  the  cylinder  and  connect  cylinder  2  (100 
c.c.  nongraduated)  to  cylinder  1  and  the  acid  wash  bottle  (see 
Fig.  15).  In  the  test  tube  containing  the  urine  place  1  c.c.  of 
amylic  alcohol  or  2  to  3  drops  of  cap ry lie  alcohol  (to  prevent 
foaming),  and  allow  about  3  to  5  c.c.  of  saturated  sodium  car- 
bonate to  run  down  the  tube  gently  (under  the  urine)  so  that 
none  of  the  ammonia  will  escape.  Place  the  test  tube  in  the  100 
c.c.  cylinder  (nongraduated)  and  then  quickly  insert  the  stopper, 
being  careful  that  the  apparatus  is  properly  connected.  Start 
the  air  from  the  suction  slowly  through  the  apparatus,  increasing 
the  speed  gradually  so  that  at  the  end  of  about  5  minutes  the  air 
current  is  as  rapid  as  the  apparatus  will  stand.  Aeration  is  com- 
plete in  15  to  20  minutes.  Disconnect  the  apparatus  and  use  cyl- 
inder 1  for  the  final  determination.  Remove  the  rubber  stopper 
from  cylinder  1  and  wash  down  the  tube  with  distilled  water  (2 
to  3  c.c.). 

We  now  develop  the  color.  In  a  50  c.c.  volumetric  flask,  pi- 
pette 5  c.c.  of  ammonium  sulphate  solution1  containing  1  mgm.  of 
nitrogen,  add  25  c.c.  distilled  water  and  20  c.c.  Nessler's  solu- 
tion2 diluted  1  to  5.  To  cylinder  1  (graduated)  containing  the  un- 

'See  footnote  2,  page  77. 
2See  footnote  3,  page  77. 


AMMONIA  83 

known,  add  15  to  25  c.c.  of  diluted  Nessler's  solution  (1  to  5), 
depending  upon  the  depth  of  color,  and  dilute  to  50  c.c.,  100  c.c., 
etc.,  depending  upon  the  depth  of  color.  The  colorimetric  read- 
ing should  be  made  at  once.  Calculation  is  made  from  the  table 
already  given  (see  page  78),  and  the  results  recorded  as  am- 
monia nitrogen. 

Example. — Suppose  the  twenty-four  hour  specimen  contains 
1500  c.c.  urine;  our  dilution  is  100,  reading  69. 

Suppose  2  c.c.  were  used  in  the  determination.  Equivalent 
from  table  is  0.70  mgm.  in  2  c.c.  urine.  Divide  by  2,  equals  0.35 
mgm.  in  1  c.c.  urine;  multiply  by  1500,  equals  525  mgm.  in  1500 
c.c.  urine  or  0.525  gram  of  ammonia  N. 


CHAPTER  XVI. 
URIC  ACID. 

Into  a  15  c.c.  conical  centrifuge  tube  pipette  2  c.c.  of  urine, 
and  add  15  drops  of  ammoniacal-silver-magnesium  mixture.1  In- 
vert the  centrifuge  tube  in  order  to  mix  the  contents  and  then  place 
the  tube  in  the  refrigerator  for  about  ten  minutes,  after  which 
centrifuge  the  tube  for  from  3  to  5  minutes,  and  then  pour  off  the 
supernatant  fluid  by  inverting  the  tube.  (The  precipitate  will 
remain  at  the  bottom.)  Wipe  the  lip  of  the  centrifuge  tube  with  fil- 
ter paper.  Volatilize  the  ammonia  by  attaching  the  mouth  of  the 
tube  to  the  suction.  We  are  now  ready  for  the  development  of 
color,  and  the  reading.  As  previously  mentioned,  we  must  again 
urge  the  beginner  to  work  as  fast  as  possible  as  the  color  may  fade 
or  turbidity  may  develop. 

Prepare  a  100  c.c.  graduated  cylinder  for  the  unknown  and  a 
50  c.c.  volumetric  flask  for  the  standard  solution.2  Then  pipette 
5  c.c.  of  uric  acid  standard  (5  c.c.  equals  1  mgm.  of  uric  acid) 
into  the  50  c.c.  volumetric  flask.  To  the  standard  solution  add 
2  drops  of  a  5%  solution  of  potassium  cyanide,  2  c.c.  of  Folin- 
Macallum3  reagent,  20  c.c.  of  saturated  sodium  carbonate,  and  in 
one  minute,  add  water  to  the  50  c.c.  mark.  (See  Plate  I  for  the 
standard  uric  acid  wedge.)  To  the  precipitate  in  the  centrifuge 
(which  is  free  from  ammonia)  add  2  drops  of  a  5%  solution  of 


nitrate  solution,  30  c.c.  of  magnesium  mixture,  and  100  c.c.  of  concentrated  ammonia.  Any 
turbidity  which  may  develop  is  removed  by  nitration.  The  magnesia  mixture  alluded  to 
is  made  as  follows:  dissolve  35  grams  of  magnesium  sulphate  and  70  grams  of  ammonium 
chloride  in  280  c.c.  of  distilled  water  and  then  add  140  c.c.  of  concentrated  ammonia. 

2For  the  preparation  of  uric  acid  standard  solution,  dissolve  9  grams  of  pure  crys- 
talline hydrogen  disodiutn  phosphate  and  1  gm.  of  dihydrogen  sodium  phosphate  in  ?00 
c.c.  to  300  c.c.  distilled  water.  Filter  and  make  up  to  about  500  c.c.  with  hot  distilled 
water.  Pour  this  warm,  clear  solution  on  200  mgms.  of  pure,  dried  uric  acid  (Kahl- 
baum)  suspended  in  a  few  cubic  centimeters  of  water  in  a  liter  flask.  Agitate  until 
completely  dissolved,  and  add  at  once  exactly  1.4  c.c.  glacial  acetic  acid.  Make  up  to 
one  liter,  mix  and  add  5  c.c.  chloroform.  Five  c.c.  of  this  solution  is  equivalent  to 
1  mgm.  of  uric  acid.  This  solution  should  be  freshly  prepared  every  two  months.  Be- 
fore weighing  out  the  200  mgms.  of  uric  acid,  it  is  well  to  dry  the  quantity  from  which 
the  measure  is  to  be  made  in  a  drying  oven  at  100°  C.  overnight. 

8For  the  preparation  of  the  Folin-Macallum  reagent,  boil  100  gms.  of  sodium  tungstate, 
20  c.c.  of  concentrated  hydrochloric  acid,  and  30  c.c.  of  85%  phosphoric  acid  in  750  c-c. 
for  two  hours  and  then  make  up  to  1000  c.c.  with  distilled  water.  In  boiling  it  is 
well  to  have  a  funnel  over  the  flask  so  as  to  prevent  undue  evaporation. 


URIC   ACID  85 

potassium  cyanide  and  shake  the  tube  so  as  to  dissolve  the  pre- 
cipitate. Add  2  c.c.  of  Folin-Macallum  reagent.  Wash  the  con- 
tents of  the  centrifuge  tube  into  the  100  c.c.  graduate  with  from 
15  to  20  c.c.  saturated  sodium  carbonate.  If  the  color  is  well 
developed,  more  carbonate  is  used ;  i.  e.,  use  the  20  c.c.  amount 
when  the  color  is  stronger  than  the  standard,  and  the  15  c.c. 
when  it  is  fainter.  The  fundamental  principle  of  these  dilutions 
in  microchemical  work  is  to  have  the  unknown  solution  weaker 
in  color  than  the  standard.  A  space  of  time  of  from  forty  to 
sixty  seconds  should  be  allowed  to  elapse  before  determining 
whether  we  are  going  to  dilute  to  50  c.c.  or  100  c.c.  Dilute  with 
distilled  water  to  50  c.c.,  100  c.c.  depending  upon  the  depth  of 
color  obtained.  The  table  for  estimation  of  uric  acid  with  the 
Hellige  colorimeter  gives  the  data  for  working  out  the  amount  of 
uric  acid  present.  (See  page  40  for  uric  acid  table.) 

Example  1. — Suppose  the  volume  of  urine  for  24  hours  is  1500 
c.c.  Dilution  is  100  c.c.  Reading  is  60.  Equivalent  from  table  is 
0.88  mgm.  in  2  c.c.  urine.  Divide  by  2,  equals  0.44  mgms.  in  1 
c.c.  urine ;  multiply  by  1500  equals  660  mgms.  in  1500  c.c.  urine 
or  0.66  gram  in  1500  c.c.  Since  uric  acid  contains  33%  nitrogen; 
the  amount  of  uric  acid  nitrogen  may  easily  be  computed  from 
this  factor  when  it  is  desired. 

Example  2. — Uric  acid  (above)  equals  0.66  gram;  33%  of  0.66 
gram  equals  0.2178  gram  of  uric  acid. 


CHAPTEE  XVII. 
CREATININE. 

Into  a  100  c.c.  volumetric  flask  or  cylinder,  pipette  2  c.c.  of 
urine.  Add  3  c.c.  of  saturated  picric  acid  and  1  c.c.  of  10%  so- 
dium hydroxide.  Mix  the  solution  thoroughly  and  allow  to  stand 
for  five  minutes.  This  is  done  to  allow  for  the  development  of 
color.  At  the  end  of  this  time  make  up  the  mixture  to  100  c.c. 
with  tap  water,  thoroughly  mix  and  read  several  times  in  the 
colorimeter,  using  normal  bichromate1  as  a  standard.  The  amount 
of  creatinine  in  2  c.c.  of  urine  is  obtained  by  ascertaining  the 
value  of  the  colorimetric  reading  in  the  Table  VII  for  the  estima- 
tion of  creatinine.  If  the  concentration  of  creatinine  in  the  urine 
is  not  such  that  the  readings  from  the  colorimeter  fall  within  the 


TABLE  VII2 


ESTIMATION  OF  CREATININE  WITH  THE  HELLIGE  COLORIMETEK 


COLORI- 

CREATININE 

COLORI- 

CREATININE 

COLORI- 

CHEATININE 

METRIC 

MOMS.  PER 

METRIC 

MOMS.  PER 

METRIC 

MGMS.  PER 

READING 

DILUTION 

READING 

DILUTION 

READING 

DILUTION 

OF    100   C.C. 

OF    100   C.C. 

OF  100  C.C. 

20 

2.46 

35 

2.13 

51 

1.78 

21 

2.43 

36 

2.10 

52 

1.76 

22 

2.41 

37 

2.08 

53 

.74 

23 

2.39 

38 

2.06 

54 

.72 

24 

2.37 

39 

2.04 

55 

.69 

25 

2.35 

40 

2.02 

56 

.67 

26 

2.33 

41 

1.99 

57 

.65 

27 

2.30 

42 

1.97 

58 

1.62 

28 

2.28 

43 

1.95 

59 

1.60 

29 

2.29 

44 

1.92 

60 

1.57 

30 

2.24 

45 

1.90 

61 

.54 

31 

2.21 

46 

1.88 

62 

.51 

32 

2.19 

48 

1.85 

63 

.48 

33 

2.17 

49 

1.83 

64 

.45 

34 

2.15 

50 

1.81 

65 

.42 

formal  bichromate  is  prepared  by  dissolving  24.55  grams  of  potassium  bichromate  in 
distilled  water,  and  making  up  to  500  c.c. 

2Myers  and  Fine:  Chemical  Composition  of  the  Blood  in  Health  and  Disease  New 
York,  1915. 


CBEATININE  87 

figures  of  the  table,  repeat  the  test,  using  larger  or  smaller  amounts 
of  urine  as  the  case  may  be. 

Example  1. — Volume  of  urine  in  twenty-four  hour  specimen  is 
1500  c.c.  Eeading  on  colorimeter  is  58.  Equivalent  on  table  is 
1.62  mgms.  in  2  c.c.  urine.  Divide  by  2  equals  0.81  mgms.  for 
1  c.c.;  multiply  by  1500,  equals  1215  mgms.  or  1.215  grams  in 
1500  c.c.  urine. 

Creatinine  contains  37.2%  of  nitrogen  and  if  the  creatinine 
nitrogen  is  desired  it  may  easily  be  calculated  from  this  factor. 

Example  2.— 37.2%  of  1.215  grams  equals  0.45198  gram  of  creat- 
inine N. 


CHAPTER,  XVIII. 
CRBATINE. 

Place  2  c.e.  of  urine  in  a  medium-sized  test  tube  and  add  2  e.c. 
of  normal  hydrochloric  acid  and  a  very  little  powdered  metallic  lead. 
Boil  the  contents  of  the  tube  nearly  to  dryness  over  a  free  flame, 
then  wash  with  as  little  water  as  possible  through  a  small  cotton  or 
glass  wool  filter  into  a  100  c.c.  volumetric  flask.  This  removes 
the  metallic  lead  which  also  reacts  with  the  picric  acid  and  alkali. 
To  the  volumetric  flask  add  3  c.c.  of  saturated  picric  acid  and  2  c.c. 
of  10%  sodium  hydroxide.  Mix  the  solution  thoroughly  and  allow 
to  stand  for  five  minutes.  At  the  end  of  this  time,  make  up  the 
mixture  to  100  c.c.  with  tap  water,  thoroughly  mix  and  read 
several  times  in  the  colorimeter,  using  the  same  standard  (normal 
bichromate)  and  table  as  for  creatinine.  The  result  obtained  is 
the  total  creatinine.  The  difference  between  the  preformed  and 
the  total  creatinine  gives  the  creatine  in  terms  of  creatinine.  By 
multiplying  this  value  by  1.16  the  weight  of  the  creatine  may  be 
obtained. 

Example. — Volume  of  urin-e  in  twenty-four  hour  specimen  is 
1500  c.c.  Eeading  on  colorimeter  is  45.  Equivalent  on  table  is 
1.90  mgms.  in  2  c.c.  urine.  Divide  by  2  equals  0.95  mgms.  in  1  c.c. 
urine;  multiply  by  1500,  equals  1.425  grams  total  creatinine  in 
1500  c.c.,  viz. : 

.Reading  =  45. 

Table  equivalent  =  1.90  +  2  =  0.95  in  1  c.c.  x  1500  =  1.425  grams  = 
total  creatinine  in  1500  c.c. 

Total  creatinine  1.425  grams  in  1500  c.c.  urine  (preformed  creat- 
inine, 1.215  grams  in  1500  c.c.  urine)  creatine  in  terms  of  creat- 
inine equal  0.210  gram  in  1500  c.c.  urine.  Multiply  0.210  by 
the  (above)  factor  1.16  which  equals  0.2436  gram  of  creatine  in 
1500  c.c.  of  urine. 


CHAPTER  XIX. 
PHENOLSULPHONPHTHALEIN. 

The  phenolsulphonphthalein  test  for  renal  function  was  de- 
vised by  Eowntree  and  Geraghty  and  depends  upon  the  injection 
into  the  tissues  of  a  dyestuff  which  is  eliminated  rather  rapidly 
by  the  normal  kidney,  and  can  be  estimated  quantitatively  in  the 
urine.  Phenolsulphonphthalein  (the  dyestuff)  is  nonirritant  to 
the  body  either  when  taken  by  mouth  or  when  injected  into  the 
tissues.  It,  therefore,  does  no  harm  to  an  already  weakened  kid- 
ney. The  patient  wrho  is  to  receive  the  injection  is  given  300  to 
400  c.c.  of  water  about  one-half  hour  previously,  in  order  to  as- 
sure a  free  flow  of  urine. 

Procedure. — Inject  1  c.c.  of  a  solution1  containing  6  mgnis.  of 
phenolsulphonphthalein  intramuscularly  in  the  lumbar  region  (the 
time  of  the  injection  being  noted) .  Allow  ten  minutes  for  the  begin- 
ning of  the  excretion  of  the  drug.  Now  collect  the  urine  for  two 
hours,  each  hour  being  kept  in  separate  bottles,  labelled  1st  hour 
and  2nd  hour.  In  other  words,  after  one  hour  and  ten  minutes, 
the  urine  is  collected  in  bottle  number  1,  and  in  two  hours  and 
ten  minutes  the  second  specimen  of  urine  is  collected  in  bottle 
number  2.  In  patients  with  obstruction  to  the  flow  of  urine  from 
the  bladder,  the  retention  catheter  is  stoppered  and  the  urine 
drawn  off  at  the  end  of  each  hour.  Other  patients  may  simply  be 
allowed  to  urinate  at  hourly  periods. 

One  c.c.  ampules  (Fig.  28)  containing  6  mgms.  of  the  dye  can 
be  purchased  at  any  reliable  drug  concern.  Fig.  29  shows  a  care- 
fully graduated  syringe  for  making  this  injection. 

To  bottle  number  1,  add  10  c.c.  of  a  W%  solution  of  sodium  hy- 
droxide and  wash  the  contents  into  a  1000  c.c.  graduate  with  tap 
water.  Then  dilute  to  1000  c.c.,  500  c.c.,  etc.,  depending  upon  the 
amount  of  dye  excreted;  i.e.,  the  more  dye  excreted,  the  greater 


^This  solution  is  prepared  by  adding  0.6  grams  of  phenolsulphonphthalein  and 
0.84  c.c.  of  2/N  sodium  hydroxide  to  enough  0.75%  sodium  chloride  solution  to  make 
100  c.c.  This  gives  the  monosodium  or  acid  salt  which  is  slightly  irritant  locally  when 
injected.  It  is  necessary  to  add  2  to  3  drops  more  2/N  sodium  hydroxide  which  changes 
the  color  to  a  bordeaux  red.  This  preparation  is  nonirritant. 


90 


BLOOD   AND   URINE    CHEMISTRY 


the  dilution.  It  is  then  read  in  the  colorimeter  with  phenolsul- 
phonphthalein  as  a  standard,  and  the  calculation  made  from  the 
Table  VIII.  (See  Plate  I  for  the  color  of  the  standard  phenol- 
sulphonphthalein  wedge.) 

To  bottle  number  2  also  add  10  c.c.  of  10%  solution  of  sodium 
hydroxide  and  wash  the  contents  into  a  1000  c.c.  graduate  with 


Fig.  28. — Phenolsulphonphthalein  ampule. 


Fig.  29. — Graduated  syringe  used  for  the 
injection  of  phenolsulphonphthalein. 


tap  water.  Then  dilute  to  1000  c.c.,  500  c.c.,  etc.,  depending  upon 
the  amount  of  dye  excreted.  Then  read  in  the  colorimeter  and 
make  the  calculation  as  above.  The  amount  of  dye  excreted  in 
both  hours  is  added  together  and  recorded.2 


^Standard  phenolsulphonphthalein  is  prepared  by  adding  10  c.c.  of  10%  sodium 
hydroxide  to  exactly  1  c.c.  of  a  solution  of  phenolsulphonphthalein  solution  containing  6 
mgms.  of  the  dye  and  making  up  to  exactly  one  liter. 


PHENOLSULPHONPHTHALEIN 


91 


Example. — First  hour  dilution  was  1000  c.c.,  reading  56,  equiva- 
lent on  table  to  45%  excretion  first  hour.  Second  hour  dilution 
was  500  c.c.,  reading  40,  equivalent  on  table  to  62%,  which  is 
divided  by  two  because  the  dilution  was  to  500  c.c.  and  the  table 
requirement  is  for  a  dilution  of  1000  c.c.  The  second  hour  is 
31  per  cent.  The  final  report  is  as  follows : 


1st  hour 
2nd  hour 

45  per  cent 
31  per  cent 

Total 

76  per  cent   (normal) 
TABLE  VIII3 

ESTIMATION  OF  PHENOLSULPHONPHTHALEIN 


PHENOL- 

PHENOL- 

PHENOL- 

PHENOL- 

SULPHON- 

SULPHON- 

SULPHON- 

SULPHON- 

COLORI- 

PHTHAL- 

COLORI- 

PHTHAL- 

COLORI- 

PHTHAL- 

COLORI- 

PHTHAL- 

METRIC 

EIN 

METRIC 

EIN 

MET-RIC 

EIN 

METRIC 

EIN 

READ- 

OUTPUT 

READ- 

OUTPUT 

READ- 

OUTPUT 

READ- 

OUTPUT 

ING 

PER  DILU- 

ING 

PER  DILU- 

ING 

PER  DILU- 

ING 

PER  DILU- 

TION OF 

TION  OF 

TION  OF 

TION  OF 

1000  C.C. 

1000  C.C. 

1000  C.C. 

1000  C.C. 

Per  Cent 

Per  Cent 

Per  Cent 

Per  Cent 

10 

94 

30 

73 

50 

52 

70 

30 

11 

93 

31 

72 

51 

50 

71 

29 

12 

92 

32 

71 

52 

49 

72 

28 

13 

91 

33 

70 

53 

48 

73 

27 

14 

90 

34 

69 

54 

47 

74 

26 

15 

89 

35 

68 

55 

46 

75 

24 

16 

88 

36 

67 

56 

45 

76 

23 

17 

87 

37 

66 

57 

44 

77 

22 

18 

86 

38 

65 

58 

43 

78 

21 

19 

85 

39 

64 

59 

42 

79 

20 

20 

84 

40 

62 

60 

41 

80 

19 

21 

82 

41 

61 

61 

40 

81 

18 

22 

81 

42 

"  60 

62 

39 

82 

17 

23 

80 

43 

59 

63 

37 

83 

16 

24 

79 

44 

58 

64 

36 

.  84 

15 

25 

78 

45 

57 

65 

35 

85 

14 

26 

77 

46 

56 

66 

34 

86 

12 

27 

76 

47 

55 

67 

33 

87 

11 

28 

75 

48 

54 

68 

32 

88 

10 

29 

74 

49 

53 

69 

31 

89 

9 

"Myers   and    Fine:      Chemical    Composition    of   the    Blood   in   Health   and   Disease,    New 
York,  1915. 

Indigo-Carmin  Test  for  Kidney  Efficiency. — This  is  the  so- 
called  indigo-carmin  test  of  Folkner  and  Joseph.     This  substance 


92  BLOOD   AND   URINE    CHEMISTRY 

comes  in  tablet  form  and  is  manufactured  by  Bruckner,  Lampe  & 
Company.  The  tablets  are  blue  and  are  soluble  in  water.  The 
solution  is  injected  intramuscularly.  These  two  observers  found 
that  the  elimination  of  indigo-carmin  begins  about  eight  to  ten 
minutes  after  its  injection.  The  original  method  of  Folkner  and 
Joseph  is  to  examine  the  bladder  through  a  cystoscope  and  observe 
the  first  appearance  of  the  blue  color  in  the  bladder.  This  is  in 
other  words  a  method  of  chromo-cystoscopy.  The  test  was  modi- 
fied by  Kapsemar  and  is  perhaps  better  carried  according  to  his 
technic:  both  ureters  are  catheterized  and  the  time  of  appearance 
of  the  blue  color  is  observed  from  either  kidney  in  this  way.  The 
indigo-carmin  is  mixed  in  the  following  manner:  five  tablets  are 
boiled  in  100  c.c.  of  distilled  water  for  three  to  four  hours.  This 
is  enough  material  for  five  injections.  Preserve  in  a  well  stoppered 
bottle  until  ready  for  use.  Inject  20  c.c.  for  a  test,  boiling  it  be- 
fore the  test  and  injecting  it  while  warm.  The  injection  is  made 
into  the  relaxed  gluteal  muscles. 

This  is  a  good  test  as  a  preliminary  measure,  but  is  only  useful 
when  positive  results  are  obtained.  If  the  blue  color  is  specifically 
delayed  on  either  side,  the  result  may  be  interpreted  as  an  indica- 
tion of  local  kidney  deficiency.  Nevertheless,  it  must  be  mentioned 
that  there  are  numerous  cases  in  which  there  has  been  well  marked 
kidney  insufficiency  and  yet  the  blue  color  appeared  promptly  on 
both  sides. 

Cryoscopy  of  Blood  and  Urine.— It  was  Koranyi  who  first 
opened  up  the  path  of  cryoscopy  in  connection  with  kidney  diag- 
nosis. The  estimation  of  the  freezing  point  of  human  blood  was 
first  used.  It  must  be  assumed  that  the  freezing  point  in  healthy 
human  blood  is  a  constant  factor.  Upon  this  point,  Koranyi  con- 
structed the  following  principle :  if  you  have  normal  kidneys,  you 
have  a  constant  freezing  point  for  blood  from  such  people.  Let 
us  assume  0.56 °C.  as  the  freezing  point  of  normal  human  blood. 
The  ease  of  freezing  is  in  proportion  to  the  number  of  molecules  in 
the  blood.  In  other  words,  the  more  molecules  present,  the  more 
difficult  it  is  to  freeze.  In  diseases  of  the  kidney  we  have  more 
molecules  in  the  blood,  ergo  the  freezing  point  of  blood  from  a 
patient  with  diseased  kidneys  is  appreciably  lowered.  This  is 
theoretically  a  good  rule  but  there  are  so  many  exceptions  that  it 
is  difficult  to  use  this  principle  in  actual  practice.  There  are  some 


PHENOLSULPHONPHTHALEIN  93 

kidney  diseases  in  which,  while  the  blood  ought  to  be  concentrated, 
there  ensues  such  a  rapid  thinning  out  that  its  freezing  point  may 
be  normal.  The  use  of  blood  cryoscopy  in  the  diagnosis  of  kidney 
disease  has  been  abandoned  by  nearly  every  one  excepting  per- 
haps Kuemmel,  of  Hamburg,  who  according  to  our  latest  informa- 
tion continues  to  use  it.  The  technic  is  not  difficult  but  there  are 
many  sources  of  mechanical  error  in  the  hands  of  the  unskilled. 
Of  more  importance  in  practical  work  is  the  cryoscopy  of  urine. 
Cryoscopy  is  a  method  of  determination  of  the  number  of  molecules 
present.  If  you  take  the  urine  from  two  sides,  one  healthy  and 
one  diseased,  you  will  find  on  the  diseased  side  a  urine  with  a  de- 
creased number  of  molecules  for  the  reason  that  the  kidney  is  not 
functionating  as  well  as  it  should  in  conditions  of  health.  It  is, 
therefore,  throwing  out  less  material,  hence  a  lessened  number  of 
molecules,  hence  freezing  of  this  urine  is  not  difficult;  therefore, 
the  freezing  point  of  urine  of  this  kind  is  higher  than  urine  from  a 
healthy  kidney.  For  instance,  if  the  left  kidney  has  a  high  freez- 
ing point,  the  right  kidney  a  lower  freezing  point,  then  it  is  the 
left  kidney  that  is  diseased.  .We  express  the  mean  average  stand- 
ard freezing  point  of  urine  by  the  large  capital  Greek  letter  Delta 
The  freezing  point  of  urine  varies  ordinarily  between  -1.3°  and 
-2.3°  C.,  the  freezing  point  of  water  being  taken  as  0°  C.  A  is  sub- 
ject to  very  wide  variations,  therefore,  its  interpretation  must  be 
taken  up  with  some  discrimination.  A  copious  drinking  of  water 
will  cause  the  A  to  have  as  high  a  value  as  -0.2°  C.  A  diet  con- 
taining much  salt  and  deficient  in  fluids  will  lower  it  to  -0.3°  C. 
Marked  variations  are  of  importance  in  reading  disease  of  the  kid- 
ney with  cryoscopy  findings.  A  concrete  example  of  the  reading  of 
cryoscopy  might  be  given  as  follows : 

Example  1. — 

Left  Kidney  Right  Kidney 

Clear  Pus 

A  =  -2.46  C.  A  =  -1.03  C. 

Diagnosis. — Pyuria  with  disturbance  of  the  right  kidney  function. 

Example  2. — 

Left  Kidney  Right  Kidney 

Clear  Pus 

A  =  -2.46  C.  A  =  -2.11 


94  BLOOD   AND   URINE    CHEMISTRY 

Diagnosis. — Pyuria,  with  disease  of  the  right  kidney,  but  the  dif- 
ference in  the  freezing  points  is  so  slight  that  it  is  not  possible  to 
absolutely  say  that  the  function  of  the  right  kidney  is  materially 
disturbed. 

Cryoscopy  is  best  carried  out  by  means  of  the  Beckman  appa- 
ratus. This  consists  of  a  heavy  battery  jar  with  a  metal  cover  with 
a  circular  hole  in  the  center.  This  jar  holds  the  freezing  mixture  by 
means  of  which  the  temperature  of  the  urine  is  lowered  and 
estimated.  A  large  glass  tube  in  the  center  serves  as  an  air-jacket 
and  is  inserted  through  the  central  hole.  Within  this  is  received 
the  small  tube  containing  the  urine  to  be  tested.  A  thermometer 
graduated  in  hundredths  of  a  degree  is  introduced  into  the  inner 
tube  and  is  held  in  place  by  means  of  a  cork  so  that  the  mercury 
bulb  is  immersed  in  the  fluid  under  examination  but  does  not  come 
in  contact  with  the  glass  surface  anywhere.  A  small  stirrer  drops 
into  the  fluid  and  is  used  to  stir  it  while  it  is  being  frozen.  Another 
stirrer  mixes  up  the  ice  and  rock  salt  mixture.  Rock  salt  one  part 
and  ice  3  parts,  makes  a  good  freezing  mixture.  Make  the  test  as 
follows:  produce  a  temperature  not  lower  than  3°  C.  in  the  freez- 
ing mixture.  Introduce  the  urine  to  be  tested  in  the  small  test 
tube,  stir  both  stirrers  so  as  to  equalize  the  temperature  slowly  and 
watch  the  column  of  mercury  in  the  thermometer  which  dips  into 
the  urine.  This  mercury  will  fall  slowly  as  freezing  occurs.  You 
will  then  observe  a  sudden  jump  in  the  mercury  column  after  it 
falls.  The  point  that  it  rises  to  after  this  jump  is  the  freezing  point. 


CHAPTER  XX. 
CHLORIDES. 

Pipette  5  c.c.  of  urine  into  a  small  evaporating  dish  and  add 
about  20  c.c.  distilled  water.  Precipitate  the  chlorides  by  the  ad- 
dition of  exactly  10  c.c.  of  standard  silver  nitrate  solution1  and 
add  2  c.c.  of  the  indicator.2  Run  in  from  a  burette  standard  am- 
monium thiocyanate  until  the  first  trace  of  yellow  shows  through- 
out the  mixture  on  stirring.  By  subtracting  the  number  of  cubic 
centimeters  required  to  exactly  precipitate  the  chlorides  from  ten 
(silver  nitrate  added)  and  multiplying  by  0.01,  the  grams  of 
sodium  chloride  in  5  c.c.  of  urine  are  obtained.  From  this  the 
total  chloride  output  for  the  twenty-four  hour  specimen  may  be 
computed.  The  twenty-four  hour  specimen  contains  1500  c.c. 
urine. 

Example. — 6.2  c.c.  standard  ammonium  thiocyanate3  used  sub- 
tracted from  10  equals  3.8  c.c.  of  silver  nitrate  (standard)  actually 
required.  Multiply  this  by  0.01  gram  (1  c.c.  of  standard  silver 
nitrate  equals  0.01  gram  of  sodium  chloride),  equals  0.038  gram  of 
sodium  chlorides  in  5  c.c.  urine.  In  1500  c.c.  urine  there  will  then 
be  300  times  0.038  gram  of  sodium  chloride  or  11.4  grams  of  sodium 
chloride  in  1500  c.c.  of  urine. 


1For  the  preparation  of  the  standard  silver  nitrate  solution,  dissolve  29.06  grams  of 
silver  nitrate  in  distilled  water  and  make  up  to  one  liter  with  distilled  water.  Each 
cubic  centimeter  of  such  a  solution  is  equivalent  to  0.01  gram  of  sodium  chloride. 

2For  the  preparation  of  the  indicator,  dissolve  100  grams  of  crystalline  ferric  am- 
monium sulphate  in  100  c.c.  of  25  per  cent  nitric  acid. 

'For  the  preparation  of  standard  ammonium  thiocyanate,  dissolve  about  13  gram's  of 
ammonium  thiocyanate  in  800  c.c.  distilled  water.  ^Titrate  this  solution  against  the 
above  standard  silver  nitrate  solution,  thus  ascertaining  the  amount  of  water  which  must 
be  added  to  the  solution  to  make  it  equivalent  to  the  silver  nitrate  solution. 


CHAPTER  XXI. 

GENERAL  ANALYSIS. 

Urine. 

Volume. — This  is  easily  measured  in  one  liter  graduates.  The 
volume  of  urine  excreted  by  normal  individuals  is  influenced 
greatly  by  the  diet,  particularly  by  the  volume  of  fluid  ingested. 
The  normal  figures  fall  within  from  1000  c.c.  to  1200  e.c. 

Pathological  conditions  which  cause  increase  in  the  output  of 
urine,  may  be  enumerated  as  follows : 

1.  Diabetes  mellitus. 

2.  Diabetes  insipidus. 

3.  Certain  diseases  of  the  nervous  system. 

4.  Contracted  kidney. 

5.  Amyloid  degeneration  of  the  kidney. 

6.  Convalescence  from  acute  diseases. 

Many  drugs,  such  as  calomel,  digitalis,  acetates,  and  salicylates, 
also  cause  an  increase  in  the  output  of  urine. 

Pathological  conditions  which  cause  decrease  in  output  of  the 
urine : 

1.  Acute  nephritis. 

2.  Diseases  of  the  heart. 

3.  Diseases  of  the  lungs. 

4.  Fevers. 

5.  Diarrhea. 

6.  Vomiting. 

Color. — The  color  of  normal  urine  varies  from  a  very  pale  yel- 
low to  a  reddish  yellow.  The  nature  and  origin  of  the  chief 
variations  in  the  urinary  color  are  set  forth  in  tabular  form  by 
Halliburton,  as  shown  in  Table  IX. 

Transparency. — Normal  urine  is  ordinarily  perfectly  clear.  On 
standing  a  few  hours  a  cloud  (nubecula)  consisting  of  mucus 
threads  epithelial  cells,  etc.,  forms.  After  a  hearty  meal  the  urine 
is  generally  turbid,  due  to  the  precipitation  of  phosphates,  and 


PLATE    III. — URINE    COLOR    REACTION'S. 


1.  Showing   Indican   Reaction. 

2.  Showing   Benzidine    Test    for    Blood. 

3.  Showing   Acetone    Reaction. 

4.  Showing  Diacetic   Acid   Reaction. 


GENERAL   ANALYSIS 


97 


TABLE  IX. 


COLOR. 


CAUSE  OF  COLORATION. 


PATHOLOGICAL 
CONDITIONS. 


Nearly  colorless 


Dark    yellow    to    brown- 
red 


Milky 

Orange 

Bed  or  reddish 


Dilution  or  diminution  of 
normal  pigments 


Increase  of  normal,  or 
occurrence  of  patho- 
logical pigments,  con- 
centrated urine 

Fat  globules 
Pus  corpuscles 


Excreted  drugs 
Hemateporphyrin 


Brown  to  brown-black 


Unchanged  hemoglobin 

Pigments  in  food  (log 
wood)  matter,  bilbu 
ries,  fuchsin. 

Hematin 

Methemoglobin 

Melanin 


Nervous  conditions,  hy- 
druria,  diabetes  insipi- 
dus,  granular  kidney 

Acute  febrile  diseases 


Chyluria 

Purulent  diseases  of  the 
urinary  tract 

Santonin,    crysophanic 
acid 

Hemorrhages,    or     hemo- 
globinuria 


. 


Small  hemorrhages 
Methemoglobinuria 
Melanotic  sarcoma 


Hydrochinol  and  catechol    Carbolic  acid  poisoning 
Jaundice 


Greenish-yellow,  greenish-    Bile-pigments 
brown  approaching  . 
black 

Dirty  green  or  blue  ;  A  dark  blue  scum  on  sur-  Cholera,  typhus ;  seen  es- 
f  ace,  with  a  blue  de-  pecially  when  the  urine 
posit,  due  to  an  excess  is  putrefying 
of  indigo-forming  sub- 
stances 

Brown  -  yellow  to  red-  Substances  contained  in 
brown,  becoming  blood-  senna,  rhubarb,  and 
red  upon  adding  alka-  chelidonium  which  are 
lies.  introduced  into  the  sys- 

tem 


will  disappear  on  the  addition  of  acetic  acid.    Permanently  turbid 
urines  generally  arise  from  pathological  conditions. 

Odor. — Normal  urine  has  a  faint  aromatic  odor.    On  standing 
a  long  time  all  urines  are  decomposed  (undergo  alkaline  fermenta- 


98  BLOOD  AND  URINE   CHEMISTRY 

tiorf)  and  have  a  very  unpleasant  ammoniacal  odor.  Certain  drugs 
(cubebs,  myrtol,  copaiba,  tolu,  saffron,  and  turpentine)  impart  a 
specific  odor  to  urine. 

Reaction. — The  urine  of  a  normal  individual  is  generally  acid  to 
litmus.  An  animal  diet  yields  an  acid  urine  while  a  vegetable  diet 
may  yield  a  neutral,  or  even  an  alkaline  urine.  The  composition  of 
the  food  taken  is  probably  the  most  important  factor  in  determin- 
ing the  reaction  of  the  urine.  The  reaction  also  varies  considerably 
according  to  the  time  of  the  day  the  urine  is  passed.  For  instance, 
for  a  variable  length  of  time  after  a  meal  the  urine  may  be  neutral 
or  even  alkaline  to  litmus.  This  change  in  reaction  is  common  to 
perfectly  healthy  individuals.  Normal  urine  becomes  alkaline  on 
standing,  owing  to  the  conversion  of  urea  into  ammonium  carbonate 
by  bacteria. 

Specific  Gravity  and  Solids. — The  specific  gravity  of  normal 
urine  varies  ordinarily  between  1.015  and  1.025.  It  may,  however, 
be  as  low  as  1.003  or  as  high  as  0.040  without  necessarily  indicat- 
ing any  pathological  condition.  For  instance,  following  copious 
water  or  beer  drinking,  the  specific  gravity  may  become  as  low  as 
1.003  or  lower.  Whereas,  on  the  other  hand  in  cases  of  excessive 
perspiration  it  may  rise  as  high  as  1.040  or  even  higher. 

In  general  (normally  and  pathologically)  the  specific  gravity  is 
inversely  proportional  to  the  volume  excreted.  In  diabetes  mellitus, 
however,  we  may  observe  a  large  volume  and  a  high  specific  gravity 
owing  to  the  sugar  contained  in  the  urine. 

For  determining  the  specific  gravity  the  urinometer  commonly 
is  used  (Fig.  30).  This  is  sufficiently  accurate  for  clinical  pur- 
poses. The  urinometer  is  always  calibrated  for  use  at  a  certain 
temperature.  If  the  specific  gravity  is  taken  at  any  other  tempera- 
ture, correction  as  given  below  must  be  made.  In  making  this 
correction,  one  unit  of  the  last  order  is  added  for  every  three 
degrees  above  the  normal  temperature  and  substracted  for  every 
three  degrees  below  the  normal  temperature. 

Example.— The  urinometer  is  calibrated  for  15°  C. 

The  specific  gravity  of  the  urine  at  18°  C.  is  1022. 

The  true  specific  gravity  at  15°  C.  would  be  1.022  +  0.001  =  1.023. 

Solids. — The  amount  of  solids  in  1000  c.c.  may  roughly  be  cal- 
culated by  means  of  Long's  coefficient,  which  is  2.6.  This  is  ob- 


GENERAL  ANALYSIS 


99 


tained  by  multiplying  the  last  two  figures  of  the  specific  gravity 
observed  at  25°  C.  by  2.6. 

Example. — The  twenty-four  hour  specimen  contains  1500  c.c. 

Specific  gravity  is  1016. 

(a)  16  x  2.6  =  41.6  grams  of  solid  matter  in  1000  c.c.  urine. 

(b)  41.6  x  1500  =  62.4  grams  of  solid  matter  in  1500  c.c.  urine. 

1000 
Toluene  is  very  satisfactory  for  preserving  urine.    This  is  simply 


/\ 


Fig.  30. — Urinometer. 

poured  into  the  specimen  so  that  the  urine  is  overlaid  with  the 
toluene. 

In  certain  pathological  conditions  it  is  desired  to  have  a  separate 
day  and  night  urine.  The  urine  voided  between  8  A.  M.  and  8  P.  M. 
is  taken  as  the  day  sample,  and  that  voided  between  8  P.  M.  and 
8  A.  M.  is  taken  as  the  night  sample. 


100  BLOOD   AND   URINE   CHEMISTRY 

Glucose. 

Qualitative  Test  for  Glucose.—  Place  about  5  c.c.  of  Benedict's 
qualitative  solution1  in  a  test  tube  and  add  8  to  10  drops  (not 
more)  of  the  urine  under  examination,  and  boil  the  mixture  vigor- 
ously for  a  minute  and  a  half.  It  is  allowed  to  cool  spontaneously. 
In  the  presence  of  dextrose,  the  entire  body  of  the  solution  will 
be  filled  with  a  precipitate,  which  may  be  red,  yellow  or  green  in 
color,  depending  upon  the  amount  of  sugar  present.  (See  Plate 
IV  for  color  of  test  for  glucose.) 

If  the  amount  of  glucose  is  small  (under  0.3$))  the  precipitate 
forms  only  on  cooling.  If  the  urine  contains  no  sugar,  the  solution 
either  remains  perfectly  clear,  or  shows  a  faint  turbidity  that  is 
blue  in  color  and  consists  of  precipitated  urates,  and  should  cause 
no  confusion.  Even  very  small  quantities  of  dextrose  (0.1%)  yield 
precipitates  of  surprising  bulk  with  Benedict's  reagent. 

Benedict's  Quantitative  Estimation  of  Glucose.2  —  The  titration 
method  of  Benedict  which  is  conceded  to  be  far  superior  to  the  older 
titration  methods  of  Fehling  and  Purdy,  is  the  method  which  is 
chosen.  This  method  gives  very  excellent  results  and  no  special  or 
expensive  apparatus  is  required.  It  is  superior  to  the  Lohnstein 
fermentation,  because  the  results  may  be  obtained  at  once  (about 
five  minutes).  It  is  also  superior  to  the  polariscope  method  in 
those  instances  when  levorotatory  substances  (as  /?-hydroxybutric 
acid)  are  present,  thus  necessitating  a  determination  both  before 
and  after  fermentation.  Place  the  urine  in  a  graduated  burette, 
pipette  25  c.c.  of  the  volumetric  solution3  into  a  Jena  flask  of  about 
150  c.c.  capacity,  and  add  5  to  10  grams  of  sodium  carbonate  and  a 
bit  of  powdered  pumice. 

Heat  the  mixture  to  boiling  on  a  piece  of  wire  gauze  with  an 
asbestos  mat  and  run  the  urine  in  rapidly  from  the  burette  until  a 
chalky  white  precipitate  begins  to  form.  (See  Fig.  31.)  Then  the 


. 
Essentials  of  Pathological  Chemistry,  1913. 


— , 


PLATE  IV. — BENEDICTS'  TEST  FOR  SUGAR. 


1.  Green — Showing    only    a    Trace    of    Sugar. 

2.  Red — Showing  a  Large  Amount  of   Sugar. 

3.  Yellow — Showing  a   Small   Amount   of    Sugar. 


GENERAL   ANALYSIS 


101 


Fig.  31. — Showing   Benedict's   method   for   the    quantitative    estimation    of   sugar. 


102  BLOOD   AND   URINE    CHEMISTRY 

urine  is  run  in  more  slowly  with  continuous  boiling,  until  the  last 
trace  of  blue  color  disappears,  indicating  the  end  point.  Chloro- 
form, if  present,  should  be  removed  by  boiling  as  it  interferes  with 
the  reaction.  Benedict  has  found  that  25  c.c.  of  the  above  copper 
solution  were  reduced  by  exactly  50  mgms.  of  glucose  or  52  mgms. 
of  levulose.  Myers  and  Fine  have  found  that  25  c.c.  of  the  above 
copper  solution  were  reduced  by  54  mgms.  of  galactose  or  67  mgms. 
of  lactose.  If  a  large  amount  of  glucose  is  present,  the  urine  should 
be  accurately  diluted  and  the  test  carried  out  in  the  same  way, 
the  final  results  being  multiplied  by  the  dilution. 

Example. — The  twenty-four  hour  specimen  of  urine  contained 
2000  c.c. 

The  amount  of  urine  required  to  reduce  25  c.c.  of  Benedict's  volu- 
metric solution  (50  mgms.  glucose)  was  10  c.c.  Therefore  10  c.c. 
of  urine  contains  50  mgms.  of  glucose.  1  c.c.  contains  10  divided 
into  50  mgms.  or  5  mgms.  2000  c.c.  contains  10,000  mgms.  or  10 
grams  of  glucose. 

If  the  above  urine  were  diluted  one-half  before  examination,  the 
result  should  be  multiplied  by  2,  or  20  grams  of  glucose. 

Albumin. 

Normal  urine  contains  a  faint  trace  of  albumin  which  is  too 
slight  to  be  detected  by  any  ordinary  method. 

Nitric  Acid  Ring-  Test  (Heller's  Test).— Place  1  c.c.  of  concen- 
trated nitric  acid  in  a  small  test  tube.  By  means  of  a  pipette  with 
a  rubber  bulb  on  one  end,  and  having  a  rugged  edge  on  the  other, 
allow  an  equal  amount  of  urine  to  run  gently  down  the  sides  of  the 
tube.  The  liquid  should  stratify,  and  if  albumin  is  present,  a  white 
ring  of  precipitated  albumin  should  appear  at  the  point  of  junc- 
ture. If  albumin  is  present  in  small  ^amounts,  the  white  ring  may 
not  appear  until  the  tube  has  been  allowed  to  stand  for  several 
minutes.  If  the  urine  is  concentrated  a  white  zone,  due  to  uric 
acid  or  urates,  may  form.  This  may  be  differentiated  from  the 
albumin  ring  by  diluting  the  concentrated  urine  with  three  or 
four  volumes  of  water.  The  experienced  worker  can  easily  differ- 
entiate between  the  uric  acid  ring  and  the  albumin  ring,  since  the 
uric  acid  ring  has  a  less  sharply-defined  upper  border,  is  generally 
broader  than  the  albumin  ring,  and  is  often  situated  above  the 


GENERAL   ANALYSIS 


103 


point  of  contact.  Various  colored  zones  due  to  bile  pigments,  etc., 
may  also  appear,  but  this  should  not  confuse  the  worker.  After  the 
administration  of  certain  drugs,  a  white  precipitate  of  resin  acids 
may  form  at  the  point  of  contact  and  may  cause  the  observer  to 
draw  wrong  conclusions.  This  ring  (if  composed  of  resin  acids) 
will  dissolve  in  alcohol,  whereas  the  albumin  ring  will  not. 

Robert's  Test  for  Albumin. — Into  a  small  test  tube  introduce 
1  c.c.  of  Robert's  reagent.3  By  means  of  a  pipette  with  a  rubber 
bulb  on  one  end,  having  a  rugged  edge  on  the  other,  allow  an  equal 
amount  of  urine  to  run  gently  down  the  sides  of  the  tube.  The 


Fig.  32. — Graduated    conical    centrifuge    tube. 

liquids  should  stratify,  and  if  albumin  is  present  a  white  zone  of 
precipitated  albumin  should  appear  at  the  point  of  juncture.  This 
test  is  slightly  more  sensitive  than  Heller's  test  and  colored  rings 
do  not  appear,  but  if  uric  acid  or  urates  are  present,  a  white  zone 
may  also  appear  and  can  be  differentiated  from  albumin  by  dilu- 
tion as  in  Heller's  test. 

Quantitative  Estimation  of  Protein  (Purdy).— Into  a  15  c.c. 
graduated  conical  centrifuge  tube  (Fig.  32)  place  10  c.c.  of  clear 
urine,  3  c.c.  of  10%  potassium  ferrocyanide,  and  2  c.c.  of  50% 
acetic  acid.  Shake  the  tube  and  set  aside  for  10  minutes  to  allow 
for  the  precipitation  of  the  albumin,  centrifuge  the  tube  for  exact- 


'Kobert's   reagent   is    prepared   by    mixing    five   parts    of    saturated   magnesium    sulphate 
and  one  part  of  concentrated  nitric  acid. 


104 


BLOOD   AND   URINE    CHEMISTRY 


ly  three  minutes,  at  1500  revolutions  per  minute,  in  an  instrument 
with  a  radius,  including  the  tubes,  of  just  6%  inches.  Then  take 
the  tube  out  of  the  centrifuge  and  the  grams  of  protein  per  liter 
are  read  off  from  the  following  table  (see  Table  X) .  If  the  amount 
of  protein  is  very  large,  the  urine  should  be  accurately  diluted. 

Example. — Precipitate  in  centrifuge  tube  is  1.25  which  is  equal 
to  2.6  grams  of  protein  per  1000  c.c.  24  hour  specimen  contains 
1500  c.c.  Multiply  2.6  by  1.5,  equals  3.9  grams  of  protein  in  1500 
c.c. 

TABLE  X 


VOLUME  OP 

DRY  WEIGHT 

VOLUME  OF 

DRY  WEIGHT 

PRECIPITATE  IN 

OF  PROTEIN 

PRECIPITATE  IN 

OF  PROTEIN 

GRADUATED  TUBE 

TO  LITER 

GRADUATED  TUBE 

TO  LITER 

0.25 

0.5 

2.75 

5.7 

0.5 

1.0 

3.0 

6.3 

0.75 

1.6 

3.25 

6.8 

1.0 

2.1  ' 

3.50 

7.3 

1.25 

2.6 

3.75 

7.8 

1.5 

3.1 

4.0 

8.3 

1.75 

3.6 

4.25 

8.9 

2.0 

4.2 

4.50 

9.4 

2.25 

4.7 

4.75 

9.9 

2.5 

5.2 

5.0 

10.4 

Acetone. 

To  10  c.c.  of  urine  in  a  test  tube  add  about  one  gram  of  am- 
monium sulphate,  2  to  3  drops  of  a  freshly  prepared  5%  solu- 
tion of  sodium  nitroprusside,  and  2  c.c.  of  concentrated  ammonium 
hydroxide  which  may  be  stratified  or  poured  on  the  mixture.  The 
presence  of  acetone  is  indicated  by  the  slow  development  of  a 
permanganate  color.  (See  Plate  III  for  acetone  color.)  The  deli- 
cacy of  this  reaction  is  1  to  20,000.  Pathologically,  the  elimination 
of  acetone  (acetonuria)  is  said  to  accompany  the  following: 

1.  Diabetes  mellitus. 

2.  Scarlet  fever. 

3.  Typhoid  fever. 

4.  Pneumonia. 

5.  Nephritis. 

6.  Phosphorous  poisoning. 

7.  Fasting. 


GENERAL  ANALYSIS  105 

8.  Grave  anemias. 

9.  Deranged  digestive  function. 
It  also  frequently  accompanies : 

1.  Autointoxication. 

2.  Chloroform  anesthesia. 

3.  Ether  anesthesia. 

It  is  believed  that  the  output  of  acetone  arises  principally  from 
the  breaking  down  of  fatty  tissues  or  fatty  food  within  the  organ- 
ism. The  acetone  elimination  has  been  shown  to  increase  when 
the  patient  is  fed  an  abundance  of  fat-containing  food  as  well  as 
during  fasting.  In  fasting,  the  decomposition  of  fat  is  increased 
due  to  the  lack  of  carbohydrate  material  and  acidosis  develops.  The 
same  is  true  with  a  carbohydrate-free  diet. 

Diacetic  Acid. 

Diacetic  acid  generally  is  excreted  under  the  same  pathological 
conditions  as  in  acetonuria,  diabetes,  fevers,  etc. 

Gerhardt's  Test.— To  about  5  c.c.  of  urine  in  a  test  tube  add 
ferric  chloride  solution,  drop  by  drop,  until  no  more  precipitate 
forms.  If  diacetic  acid  is  present,  a  violet-red  or  Bordeaux-red  is 
produced.  A  variety  of  drugs  or  their  derivatives  will  give  a  posi- 
tive reaction  when  present  in  the  urine  so  that  a  positive  result  in- 
dicates the  possible  presence  of  diacetic  acid.  If  confusion  due  to 
drugs  is  suspected,  boil  the  red  solution  for  2  to  3  minutes.  If  the 
color  is  due  to  diacetic  acid,  it  should  disappear  during  boiling 
and  not  reappear  on  cooling.  (See  Plate  III  for  diacetic  acid 
color.)  / 

Indican. 

Normally,  5  to  20  milligrams  of  indican  are  eliminated  in  24 
hours.  This  amount  is  greatly  increased  in  conditions  of  excessive 
intestinal  putrefaction.  Of  the  putrefaction  products,  the  indole, 
skatole,  phenol  and  paracresol  appear  in  part  in  the  urine  as 
ethereal  sulphuric  acids,  whereas  the  oxyacids  pass  unchanged  into 
the  urine.  The  potassium  indoxyl  sulphate  content  in  the  urine 
is  a  rough  indicator  of  the  extent  of  the  putrefaction  within  the  in- 
testine. The  portion  of  the  indole  which  is  excreted  in  the  urine 
is  subjected  to  a  series  of  changes  within  the  organism  and  is 
eliminated  as  indican. 


106  BLOOD   AND   URINE    CHEMISTRY 

Obermayer's  Test. — Shake  about  10  c.c.  of  faintly  acid  urine  with 
about  0.1  gram  of  basic  lead  acetate,  and  filter.  To  the  clear  filtrate 
in  a  test  tube  add  an  equal  volume  of  Obermayer's  reagent,4  and 
about  3  c.c.  to  5  c.c.  of  chloroform.  Place  the  thumb  over  the 
mouth  of  the  tube  and  shake  vigorously.  On  standing  a  few 
minutes  the  chloroform  will  settle  and  it  will  assume  a  blue  color, 
if  indican  is  present.  (See  Plate  III  for  color  of  indican  test.) 
The  intensity  of  the  color  will  vary  with  the  amount  of  indigo 
blue  which  has  been  brought  into  solution  by  the  chloroform.  Nor- 
mally, the  chloroform  should  assume  only  a  faint  blue  color.  In 
other  words,  normal  urine  contains  a  trace  of  indican.  Qualita- 
tively, the  depth  of  blue  color  may  be  taken  as  indicating  the  de- 
gree of  indicanuria,  i.  e.,  a  deep  blue  indicates  a  large  amount  of 
indican  present. 

Phosphates. 

The  total  output  of  phosphoric  acid  is  extremely  variable,  but  the 
average  excretion  as  P205  in  24  hours  is  about  2.5  grams. 
.  Pathological  conditions  in  which  the  excretion  of  phosphates  is 
increased : 

1.  Diffuse  periostitis. 

2.  Osteomalacia. 

3.  Rickets. 

4.  Copious  water  drinking. 

Some  investigators  claim  that  the  excretion  of  phosphates  is  also 
increased  in  the  following: 

1.  Early  stages  of  pulmonary  tuberculosis. 

2.  Diseases  which  are  accompanied  by  an  extensive  decomposi- 
tion of  nervous  tissue. 

3.  Acute  yellow  atrophy  of  the  liver. 

4.  After  sleep  induced  by  potassium  bromide  or  chloral  hydrate. 
Pathological  conditions  in  which  the  excretion  of  phosphates  is 

decreased : 

1.  Acute  infectious  diseases. 

2.  Pregnancy  (in  the  period  during  which  the  fetal  bones  are 
forming) . 

3.  Diseases  of  the  kidney  (due  to  nonelimination) . 

"Obermayer's  reageht  is  prepared  by  dissolving  about  3  grams  of  ferric  chloride  in  one 
liter  of  concentrated  hydrochloric  acid. 


GENERAL    ANALYSIS 


107 


Test  for  Phosphates. — Place  50  c.c.  of  urine  in  a  beaker  or  Er- 
lenmeyer  flask,  add  5  c.c.  of  accessory  solution,5  and  heat  to  the 
boiling  point.  A  standard  solution  of  uranium  nitrate6  is  then  run 
from  a  burette  into  the  hot  solution  (drop  by  drop)  until  the  pre- 
cipitate ceases  to  form.  A  drop  of  the  mixture  brought  into  con- 
tact with  a  drop  of  10%  solution  of  potassium  ferrocyanide  on  a 
porcelain  tablet  (Fig.  33)  should  produce  a  brownish-red  color. 
If  this  color  does  not  appear,  more  standard  uranium  nitrate  solu- 


Fig.   33. — Porcelain  tablet  for  the  determination  of  phosphates. 

tion  should  be  added,  i.e.,  until  the  brownish-red  color  appears. 
The  reading  on  the  burette  is  taken  and  is  calculated  as  follows: 

Multiply  the  reading  on  the  burette  by  0.005  to  obtain  the  grams 
of  P205  in  50  c.c.  of  urine. 

Example. — 24  hour  specimen  contains  1500  c.c.  urine. 

Reading  on  burette  is  10.2. 

10.2  x  0.005  =  0.051  gram  of  P203  in  50  c.c.  urine. 

0.051  x  30  =  1.53  grams  of  P205  in  1500  c.c.  urine. 

Bile. 

When  bile  pigments  are  found  in  urine  it  may  be  regarded  as  a 
pathological  condition.  A  urine  containing  bile  is  yellowish-green 


6For  the  preparation  of  the  accessory  solution,  dissolve  100  gms.  of  sodium  acetate  in 
about  800  c.c.  distilled  water,  then  add  100  c.c.  30%  acetic  acid  to  the  solution  and  make 
up  to  one  liter  with  distilled  water. 

6For  the  preparation  of  uranium  nitrate,  dissolve  44.8  grams  of  uranium  nitrate  in 
about  900  c.c.  of  distilled  water.  Titrate  this  solution  with  a  standard  phosphate  solu- 
tion containing  0.005  gram  of  P2O5  per  cubic  centimeter.  This  standard  phosphate  is 
prepared  by  dissolving  14.721  grams  of  pure  air-dry  sodium  ammonium  phosphate 
(NaNH4HPO4+4  H2O)  in  distilled  water  and  making  up  to  one  liter.  The  amount  of 
water  to  be  added  to  the  uranium  nitrate  solution  so  that  1  c.c.  will  be  equivalent  to 
0.005  gram  of  P2O5  can  be  calculated. 


108  BLOOD   AND   URINE    CHEMISTRY 

to  brown  in  color  and  when  shaken  foams  readily,  the  foam  being 
light  yellow  in  color. 

Tests  for  Bile. — The  shaking  of  the  urine  and  observation  of 
the  color  of  the  foam  is  a  valuable  test  for  the  presence  of  bile 
pigments. 

Gmelin's  Test. — Place  1  c.c.  of  concentrated  nitric  acid  in  a 
small  test  tube.  By  means  of  a  pipette  with  a  rubber  bulb  on  one 
end,  having  a  rugged  edge  on  the  other,  allow  an  equal  amount  of 
urine  to  run  gently  down  the  sides  of  the  tube.  The  liquid  should 
stratify  and  if  bile  is  present,  various  colored  rings  (green,  blue, 
violet,  red,  and  reddish-yellow)  will  be  noted  at  the  point  of  contact. 

Smith's  Test. — Place  1  c.c.  of  dilute  tincture  of  iodin  (1  to  10) 
in  a  small  test  tube.  By  means  of  a  pipette  with  a  rubber  bulb  at 
one  end,  having  a  rugged  edge  at  the  other,  allow  an  equal  part  of 
urine  to  run  gently  down  the  sides  of  the  tube.  The  liquids  should 
stratify  and  if  bile  is  present  a  green  ring  will  be  noted  at  the 
point  of  contact. 

Blood. 

Benzine  Test. — To  about  3  c.c.  of  a  saturated  solution  of  ben- 
zidine  in  glacial  acetic  acid  add  an  equal  volume  of  hydrogen  perox- 
ide (3%)  and  1  or  2  c.c.  of  the  urine  to  be  examined.  Shake  the 
tube  and  in  the  presence  of  blood  a  blue  or  green  color  will  de-. 
velop.  See  Plate  III  for  the  color  of  the  blood  test,  A  control 
should  always  be  made  using  water  instead  of  urine.  This  is  a  very 
sensitive  test. 

Guaiac  Test. — Place  about  5  c.c.  of  urine  in  a  test  tube  and  add 
freshly  prepared  alcoholic  solution  of  guaiac  (1  to  60)  until  the 
whole  becomes  turbid.  Then  add  hydrogen  peroxide  or  old  turpen- 
tine until  a  blue  color  appears  (if  blood  is  present).  This  test 
gives  positive  results  if  old  or  partly  putrefied  pus  is  present,  even 
before  turpentine  or  peroxide  of  hydrogen  is  added. 

Fresh  pus  gives  positive  results  upon  the  addition  of  hydrogen 
peroxide. 

The  above  test  gives  a  positive  reaction  before  and  after  boiling 
(15  to  20  seconds)  if  blood  is  present.  Pus  does  not  react  after 
boiling. 

Milk,  pus,  saliva,  etc.,  give  positive  reactions  with  the  guaiac  test, 
but  do  not  respond  after  boiling  from  15  to  20  seconds. 


CHAPTER  XXII. 
MICKOSCOPIC  ANALYSIS  OF  URINARY  SEDIMENTS. 

The  value  of  the  microscopic  examination  of  the  urinary  sedi- 
ments of  pathological  urines  is- of  very  great  importance  from  the 
diagnostic  point  of  view.  The  sediments  may  be  divided  into  two 
classes  (a)  organized,  and  (b)  unorganized  sediments. 

Preparation  of  Sediment. — Pour  the  urine  under  examination 
into  a  conical  centrifuge  tube  (Fig.  34B)  and  centrifuge  (Fig.  34A) 


Fig.  34A.  —  Centrifuge. 


Fig.  34B.  —  Conical    centrifuge    tube. 


for  from  five  to  ten  minutes.  At  the  end  of  this  time,  take  the 
tube  out  of  the  centrifuge  and  introduce  a  pipette  into  the  bottom 
of  the  tube,  a  finger  being  placed  over  the  upper  opening  of  the 
pipette  so  as  not  to  allow  any  urine  to  enter  the  pipette  while  it  is 
being  placed  to  the  bottom  of  the  tube.  When  the  pipette  touches 
the  bottom,  the  finger  is  removed  and  the  deposit  will  flow  up  into 
the  pipette.  Again  close  the  upper  end  of  the  pipette  and  place  a 
drop  of  the  sediment  on  a  clean  slide.  Then  place  a  cover-glass  over 


110  BLOOD   AND   URINE    CHEMISTRY 

the  sediment.  In  our  laboratories  we  first  examine  the  sediment 
under  the  low  power,  care  being  taken  that  a  good  deal  of  the  light 
is  shut  off.  Casts  are  not  easily  seen  in  the  presence  of  much  light. 
The  sediment  is  then  examined  under  the  high  power  dry  lens.  In 
this  way  any  suspicious  elements  under  the  low  power  may  be 
clearly  seen  under  the  high  power.  When  the  urine  is  to  be  ex- 
amined for  bacteria,  etc.,  the  sediments  are  stained  (see  following 
chapter)  and  examined  under  the  oil-immersion  lens. 


Organized  Sediments.  — 

granular. 

hyaline. 

epithelial. 

1.  Casts 

blood. 

fatty. 

waxy. 

pus. 

2.  Cylindroids. 

3.  Epithelial  cells. 

4.  Leucocytes   (pus  cells). 

5.  Erythrocytes. 

6.  Spermatozoa. 

7.  Urethral  filaments. 

8.  Tissue  debris. 

'  9.  Animal  parasites. 

10.  Fibrin. 

11.  Microorganisms. 

12.  Foreign  substances  due  to  contamination. 

CASTS. — Casts  are  moulds  of  uriniferous  tubules.  They  vary 
considerably  in  size,  but  nearly  always  have  parallel  sides  and 
rounded  ends.  The  finding  of  casts  generally  indicates  some  kid- 
ney disorder,  especially  if  accompanied  by  albumin  in  the  urine. 

Granular  Casts. — The  granular  material  generally  consists  of  al- 
bumin, epithelial  cells,  fat,  or  disintegrated  erythrocytes  or  leu- 
cocytes. The  character  of  the  cast  varies  according  to  the  size  and 
nature  of  the  granules,  i.  e.,  finely  granular  casts  or  coarsely  granu- 
lar casts  (Figs.  35  A  and  5). 


MICROSCOPIC   ANALYSIS   OF   URINARY   SEDIMENTS 


111 


Fig.  35/1. — Granular  casts.     (After  Hawk.) 


Fig.  35B. — Granular  casts.     (After  Peyer.) 

Hyaline  Casts. — Hyaline  casts  are  pale  transparent,  homogeneous, 
and  are  the  most  difficult  form  of  renal  casts  to  detect  under  the 
microscope.  They  are  common  to  all  kidney  disorders  (Fig.  36). 

Epithelial  Casts. — Epithelial  casts  bear  upon  their  surface 
epithelial  cells  and  are  found  in  large  numbers  in  acute  nephritis 
(Figs.  37  A  and  B). 

Blood  Casts. — The  appearance  of  these  casts  in  the  urine  de- 
notes acute  diffuse  nephritis,  acute  congestion  of  the  kidney,  or 
renal  hemorrhage  (Fig.  38a). 


112 


BLOOD   AND   URINE    CHEMISTRY 


Fig.  36.— Hyaline  casts.     (After  Hawk.) 


Fig.  37A.— Epithelial   casts.      (After   Hawk.) 


Fig.  37B.— Epithelial   casts.      (After   Hawk.) 


MICROSCOPIC   ANALYSIS   OF   URINARY   SEDIMENTS 


113 


Fig.  38. — (a)  Blood  casts  (yellow  in  color);   (fc)  Pus  casts.     (After  Hawk.) 


Fig.  39. — Fatty  casts.      (After  Peyer.) 

Fatty  Casts. — The  appearance  of  these  casts  denotes  fatty  de- 
generation of  the  kidney  and  are  characteristic  of  subacute  and 
chronic  inflammation  of  the  kidney  (Fig.  39). 

Waxy  Casts. — Waxy  casts  do  not  appear  in  any  particular  form 
of  nephritis,  but  are  rather  common  in  amyloid  disease. 


114 


BLOOD   AND   URINE    CHEMISTRY 


Fig.  40/4.— Cylindroids.     (After  Peyer.) 


Fig.  40B.— Cylindroids.      (After  v.   Jaksch.) 


Pus  Casts. — The  surfaces  of  these  casts  are  covered  with  pus  or 
leucocytes.  Pus  casts  are  rare  and  indicate  renal  suppuration 
(Fig.  38b). 

CYLINDROIDS. — Cylindroids  are  often  mistaken  for  casts  but  are 
flat  and  smaller  in  diameter  than  casts.  These  cylindroids  or  false 


MICROSCOPIC    ANALYSIS    OF    URINARY    SEDIMENTS 


115 


casts  may  become  coated  with  urates  and  be  mistaken  for  granular 
casts.  These,  however,  disappear  on  warming.  Cylindroids  have 
no  particular  significance  because  they  are  found  in  normal  and 
pathological  urine  (Figs.  40  A  and  B). 


Fig.  42. — Human  spermatozoa.      (After  Hawk.) 

ERYTHROCYTES. — These  appear  in  the  urine  as  the  normal  bicon- 
cave or  crenated  erythrocyte  (Fig.  41). 

The'  pathological  conditions  in  which  erythrocytes  are  found  in 
the  urinary  sediment,  are  as  follows : 

1.  Hemorrhage  of  the  kidney. 

2.  Hemorrhage  of  the  urinary  tract. 


116  BLOOD   AND   URINE    CHEMISTRY 

3.  Hemorrhage  from  congestion. 

4.  Traumatic  hemorrhage. 

5.  Hemorrhagic  diathesis. 

SPERMATOZOA. — Spermatozoa  may  appear  after  coitus  or  in  the 
following  pathological  conditions  (Fig.  42)  : 

1.  Diseases  of  the  genital  organs. 

2.  Nocturnal  emissions. 

3.  Epileptic  and  other  convulsive  attacks. 

4.  They  may  or  may  not  be  motile.     They  have  an  oval  body 
and  a  long,  delicate  tail. 

URETHRAL  FILAMENTS. — These  peculiar  thread-like  bodies  may 
be  found  in  normal  urines,  and  also  in  the  following  pathological 
conditions : 

1.  Acute  gonorrhea. 

2.  Chronic  gonorrhea. 

3.  Urethrorrhea. 

These  filaments  are  generally  macroscopical.  The  first  morning 
urine  is  best  to  be  examined  for  filaments. 

TISSUE  DEBRIS. — The  finding  of  fragments  of  tissue  may  some- 
times throw  some  light  upon  a  pathological  condition.  These  tis- 
sues may  be  found  in  the  following  pathological  conditions: 

1.  Tubercular  affections  of  the  kidney. 

2.  Tubercular  affections  of  the  urinary  tract. 

3.  Tumor  of  the  kidney. 

4.  Tumor  of  the  urinary  tract. 

It  is  necessary,  however,  to  make  a  histological  examination  of 
these  tissue  fragments  before  coming  to  a  final  conclusion  as  to 
their  origin. 

FIBRIN. — Fibrin  clots  are  occasionally  found  in  the  sediments 
of  urines,  following  hematuria. 

FOREIGN  SUBSTANCES,  DUE  TO  CONTAMINATION. — Care  should  be 
taken  that  such  substances  as  starch  granules,  hair,  fat,  sputum, 
muscle  fibers,  particles  of  food,  fibers  of  silk,  wool,  linen,  etc.,  are 
not  mistaken  for  any  of  the  true  conditions  in  urine. 

Unorganized  Sediments. — 

1.  Ammonium  magnesium  phosphate  (triple  phosphate). 

2.  Calcium  oxalate. 

3.  Calcium  phosphate. 


MICROSCOPIC   ANALYSIS    OF   URINARY   SEDIMENTS 


117 


4.  Calcium  sulphate. 

5.  Calcium  carbonate. 

6.  Uric  acid. 

7.  Urates. 

8.  Cystine. 

9.  Cholesterol. 

10.  Hippuric  acid. 

11.  Leucine,  tyrosine. 


Fig.  43. — "Triple  Phosphate."     (After  Ogden.) 

AMMONIUM  MAGNESIUM  PHOSPHATE  (TRIPLE  PHOSPHATE). — This 
compound  (Fig.  43)  is  characteristic  when  the  urine  has  under- 
gone alkaline  fermentation,  either  before  or  after  being  voided,  and 
crystallized  in  two  forms,  i.  e.,  prisms  and  the  star-shaped  leathery 
crystals.  These  crystals  may  rarely  appear  in  amphoteric  or  faintly 
acid  urines,  provided  the  ammonium  salts  are  present  in  large 
enough  quantity. 


118 


BLOOD   AND   URINE    CHEMISTRY 


The  pathological  conditions  in  which  these  crystals  are  fre- 
quently abundant,  are  as  follows: 

1.  Retention  of  urine  in  the  bladder. 

2.  Paraplegia. 

3.  Chronic  cystitis. 

4.  Enlarged  prostate. 

5.  Chronic  pyelitis. 

CALCIUM  OXALATE.— These  crystals  (Fig.  44)  appear  in  the  uri- 
nary sediment  in  at  least  two  forms,  i.  e.,  octahedral  type  and  the 
dumb-bell  type.  They  may  be  found  in  acid,  neutral  or  alkaline 


urines,  but  are  most  frequently  found  in  acid  urines.  Calcium  oxa- 
late  crystals  are  found  in  normal  urines,  but  are  increased  in  the 
following  pathological  conditions: 

1.  Diabetes  mellitus. 

2.  Organic  diseases  of  the  liver. 

3.  Diseases  of  the  heart. 

4.  Diseases  of  the  lungs. 

These  crystals  are  found  in  the  urine  after  the  ingestion  of  to- 
matoes, garlic,  rhubarb,  oranges,  asparagus,  etc. 

CALCIUM  PHOSPHATE  (STELLAR  PHOSPHATE). — Calcium  phos- 
phate (Fig.  45)  may  occur  in  the  urine  in  the  amorphous,  granu- 
lar or  crystalline  form  and  are  wedge-shaped  and  often  appear  in 
rosette  arrangements.  These  crystals  are  sometimes  mistaken  for 
sodium  urate,  but  may  be  distinguished  from  the  latter  by  dis- 


MICROSCOPIC   ANALYSIS   OF   URINARY   SEDIMENTS 


119 


solving  them  in  acetic  acid.    Acetic  acid  will  readily  dissolve  the 
phosphate,  whereas  the  urate  is  much  less  soluble. 

The  pathological  conditions  in  which  calcium  phosphate  crys- 
tals are  abundant  are  as  follows: 

1.  Retention  of  urine  in  the  bladder. 

2.  Paraplegia. 

3.  Chronic  cystitis. 

4.  Enlarged  prostate. 

5.  Chronic  pyelitis. 


Fig.  45. — Calcium  phosphate  crystals. 

CALCIUM  SULPHATE. — These  crystals  (Fig.  46)  are  very  rarely 
seen  and  are  only  found  in  acid  urines.  Calcium  sulphate  crys- 
tals appear  as  long,  thin,  colorless  needles  or  prisms  and  may  be 
mistaken  for  calcium  phosphate.  They  are  readily  distinguished, 
however,  by  the  fact  that  calcium  sulphate  crystals  are  readily 
soluble  in  acetic  acid.  These  crystals  (calcium  sulphate)  are  of 
practically  no  clinical  importance. 

CALCIUM  CARBONATE. — Calcium  carbonate  crystals  (Fig.  47)  al- 
most always  appear  in  alkaline  urine,  but  may  occur  in  ampho- 
teric  or  faintly  acid  urine.  They  very  frequently  appear  in  the 


120 


BLOOD   AND   URINE    CHEMISTRY 


Fig.  46.— Calcium  sulphate.      (After  Hensel  and  Weil.) 


Fig.  47. — Calcium  carbonate  crystals.      (After   Hawk.) 


MICROSCOPIC   ANALYSIS   OF   URINARY   SEDIMENTS 


121 


dumb-bell  shape  and  can  be  differentiated  from  calcium  oxalate, 
inasmuch  as  they  dissolve  in  acetic  acid,  with  the  evolution  'of 
carbon  dioxide  gas,  while  calcium  oxalate  remains  unchanged  in 
acetic  acid. 

URIC  ACID. — Uric  acid  crystals  (Fig.  48)  appear  in  acid  urines 
in  the  following  forms : 

1.  Wedge-shaped. 

2.  Dumb-bells. 

3.  Rhombic  prisms. 

4.  Whetstones. 

5.  Prismatic  rosettes. 

6.  Irregular  or  hexagonal  plates. 


Fig.  48. — Uric  acid  crystals. 

These  crystals  generally  appear  in  the  urine  colored  brownish- 
red,  although  occasionally  they  can  be  seen  perfectly  colorless. 
The  presence  of  uric  acid  in  the  urinary  sediment  does  not  neces- 
sarily indicate  any  pathological  condition;  neither  does  it  mean 
that  the  uric  acid  content  of  the  urine  is  increased. 

The  pathological  conditions  in  which  uric  acid  is  found  in  the 
sediment,  are  as  follows : 

1.  Gout. 


122 


BLOOD   AND   URINE    CHEMISTRY 


Fig.  49. — Acid   sodium   urate   crystals.      (After   Hawk.) 


Fig.  50.— Ammonium  urate  crystals.     (After  Peyer.) 


MICROSCOPIC    ANALYSIS    OF    URINARY   SEDIMENTS 


123 


2.  Acute  febrile  conditions. 

3.  Chronic  interstitial  nephritis. 

URATES. — This  may  appear  as  ammonium,  calcium,  magnesium, 
potassium,  and  sodium  urate.  The  calcium,  magnesium,  potassium, 
and  sodium  urates  appear  in  acid  urines,  while  the  sediment  of 
ammonium  urate  appears  in  neutral,  alkaline,  or  acid  urines. 

Sodium  Urate. — Sodium  urate  (Fig.  49)  may  be  amorphous  or 
crystalline.  When  crystalline  it  appears  in  sheaves  or  clusters 
of  colorless  needles. 

Ammonium  Urate  generally  appears  in  the  burr-like  form  of 
the  "thorn-apple"  (Fig.  50),  which  appears  to  be  balls  with 
spicules  attached. 


Fig.   51. — Cholesterol  crystals.      (After  Hawk.) 

The  pathological  conditions  in  which  urates  may  appear  in  the 
urine  are  somewhat  similar  to  those  of  uric  acid. 

CYSTINE. — Cystine  is  rarely  found  in  urinary  sediments  and 
appears  in  the  form  of  thin,  colorless,  hexagonal  plates.  It  is 
insoluble  in  water,  alcohol  and  acetic  acid,  and  soluble  in  minerals, 
hydrochloric  acid,  alkalies,  and  especially  in  ammonia. 

CHOLESTEROL. — Cholesterol  crystals  are  very  rarely  found  in 
urinary  sediments  and  ordinarily  crystallize  in  regular  and  ir- 
regular colorless  plates  which  are  transparent  (Fig.  51).  They 


124 


BLOOD   AND   URINE    CHEMISTRY 


may  occasionally  be  found  as  a  film  on  the  surface  of  the  urine  in- 
stead of  in  the  sediment. 

The  pathological  conditions  in  which  cholesterol  crystals  have 
been  found  in  the  urine,  are  as  follows: 

1.  Cystitis. 

2.  Pyelitis. 

3.  Chyluria. 

4.  Nephritis.       • 

HIPPUBIC  ACID. — This  is  very  rarely  found  in  urinary  sedi- 
ments. The  crystals  appear  as  needles  or  prisms  which  are  gener- 
ally pigmented  in  the  manner  of  uric  acid  crystals. 


Fig.  52. — Hippuric  acid   crystals. 

Hippuric  acid  crystals  (Fig.  52)  are  more  soluble  in  water 
and  ether  than  uric  acid  crystals.  These  crystals  have  practi- 
cally no  clinical  significance. 

LEUCINE  AND  TYROSINE. — These  almost  always  appear  in  the 
urine  together.  They  may  be  in  solution  or  as  a  sediment.  Leu- 
cine  crystallizes  in  characteristic  spherical  masses  and  is  highly 
refractive  (Fig.  53). 

The  pathological  conditions  in  which  leucine  and  tyrosine  have 
been  found,  are  as  follows : 

1.  Acute  yellow  atrophy  of  the  liver. 

2.  Acute  phosphorous  poisoning. 


MICROSCOPIC   ANALYSIS   OP   URINARY   SEDIMENTS  125 

3.  Cirrhosis  of  the  liver. 

4.  Severe  cases  of  typhoid  fever. 

5.  Severe  cases  of  smallpox. 

6.  Leukemia. 

Urinary  Calculi. — Urinary  calculi  are  solid  masses  of  urinary 
sediment  and  are  formed  in  some  part  of  the  urinary  tract.  The 
smaller  calculi,  termed  sand  or  gravel,  generally  arise  from  the 
kidney  or  the  pelvic  portion  of  the  kidney.  The  large  calculi  are 
generally  formed  in  the  bladder.  Calculi  are  divided  into  two  gen- 
eral classes  [according  to  their  composition,  i.  e.,  simple  (made  up 


Fig.  53. — Crystals  of  impure  leucine.     (After  Ogden.) 

of  a  single  constituent)  and  compound  (made  ifp  of  two  or  more 
constituents)  ] . 

URIC  ACID  AND  URATE  CALCULI. — These  stones  are  always  col- 
ored and  vary  from  a  pale  yellow  to  a  brownish-red. 

PHOSPHATIC  CALCULI. — These  concretions  consist  principally  of 
"triple  phosphate"  and  other  phosphates  of  the  alkaline  earths, 
with  very  frequent  admixtures  of  urates  and  oxalates  (Hawk). 

CALCIUM  OXALATE  CALCULI. — This  is  rather  difficult  to  crush 


126  BLOOD   AND   URINE    CHEMISTRY 

and  generally  occurs  in  two  forms,  the  small  (hemp  seed  calculus) 
and  the  medium  or  the  large  (mulberry  calculus) . 
The  following  calculi  are  rarely  found: 

1.  Calcium  carbonate   (extremely  rare). 

2.  Cystine  (rare). 

3.  Xanthine  (more  rare  than  the  cystine  type). 

4.  Urostealith    (extremely   rare). 

5.  Fibrin  (rare). 

6.  Cholesterol  (extremely  rare). 

7.  Indigo  (extremely  rare — only  two  cases  have  been  reported). 

In  examining  the  urinary  calculi  chemically,  the  most  valu- 
able data  are  obtained  by  examining  each  of  the  concentric  lay- 
ers separately.  One  should  saw  the  calculi  through  the  nucleus 
and  separate  the  various  layers.  Enough  material  may  also  be 
obtained  by  scraping  enough  powder  from  each  layer  to  carry 
out  the  examination.  If  the  latter  is  adapted,  the  layers  should 
not  be  separated. 

Murexide  Test. — To  a  small  amount  of  unknown  in  a  small 
evaporating  dish  add  2  to  3  drops  of  concentrated  nitric  acid. 
Evaporate  to  dryness  over  a  water-bath.  If  uric  acid  is  present, 
a  red  or  yellow  residue  remains  which  turns  purplish  red  after 
cooling  the  dish  and  adding  a  drop  of  very  dilute  ammonium 
hydroxide.  The  color  is  due  to  the  formation  of  ammonium  pur- 
purate  or  murexide.  If  potassium  hydroxide  is  used  instead  of 
ammonium  hydroxide  a  purplish  violet  color  due  to  the  pro- 
duction of  the  potassium  salt  is  obtained.  The  color  disappears 
upon  warming;  with  certain  related  bodies  (purine  bases)  the 
color  persists  under  these  conditions. 

The  following  is  a  scheme  proposed  by  Heller  for  the  chem- 
ical examination  of  urinary  calculi  and  will  be  found  very  use- 
ful in  ^determining  their  composition.  Reduce  ,th'e  Calculus  to 
powder  and  proceed  as  follows : 


MICROSCOPIC    EXAMINATION    OF    URINARY    SEDIMENT  127 

TABLE  XI 
ON  HEATING  THE  POWDER  ON  PLATINUM  FOIL,  IT 


DOES   NOT   BURN 

DOES   BURN 

The  Powder  when  Treated  with 
HCI 

With  Flame 

Without 
Flame 

Does  not  effervesce 

JJ-J 

CT  P 

m 

•73   tT'U 

111 

3    3-3 

The    Powder 
gives    the 
Murexide 
Test* 

The    Powder    gently 
heated,  then  treated 
with  HCI 

tlP 

I'l 
5" 

O    •?    P 

^"'Sji* 

The    Powder 
when    treated 
with  KOH 

The  powder  when 
moistened  with  a 
little  KOH 

fill 

li 

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1 

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S-  2-  c 

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*See  page  126  for  murexide  test. 


CHAPTER  XXIII. 

THE  STAINING  OF  BACTERIA  IN  URINE. 

Freshly  voided  urine  from  normal  persons  is  free  from  bac- 
teria, but  on  standing  it  becomes  loaded  with  saprophytic  organ- 
isms. Fungi  are  prone  to  develop  quickly  in  diabetic  urine. 
Actinomycosis  of  the  genitourinary  tract  embodies  the  finding  of 
the  actinomyces  in  the  urine.  In  general  aspergillosis,  the  As- 
pergillus  fumigatus  appears  in  the  urine.  Of  the  bacteria  to  be 
met  with  in  urine  in  pathological  states,  we  must  consider  the 
Bacillus  typhosus  which  is  found  in  at  least  thirty  per  cent  of 
all  cases  of  typhoid  fever.  Again  we  may  find  the  streptococcus, 
the  staphylococcus,  the  gonococcus,  and  the  glanders  bacillus. 
These  are,  of  course,  met  with  in  specific  infections.  In  nephritis 
of  children  we  are  apt  to  find  the  streptococcus  and  the  Bacillus 
coli  communis.  The  latter  organism  is  frequently  found  in  the 
urine  from  cases  of  acute  cystitis  and  pyelitis.  The  Staphylococcus 
pyogenes  albus  and  aureus  are  seen  in  cases  of  acute  cystitis  and, 
occasionally  the  Bacillus  pyocyaneus. 

The  organism  that  is  possibly  the  most  important  one  from 
the  standpoint  of  diagnosis  of  urinary  sediment  is  the  Bacillus 
tuberculosis.  Tuberculosis  of  the  genitourinary  tract  is  not  an 
uncommon  condition.  Thanks  to  the  exceedingly  careful  work 
of  the  modern  urologist,  this  disease  is  frequently  recognized  in 
time  to  save  life,  inasmuch  as  the  Great  White  plague  in  this 
locality  is,  almost  strictly  speaking,  a  surgical  condition.  Prompt 
diagnosis  and  prompt  extirpation  of  a  tuberculous  kidney  will 
often  result  in  a  success.  The  diagnosis  of  tuberculosis  from 
the  urinary  sediment  is,  therefore,  extremely  important.  Whether 
the  specimen  represents  a  catheterized  ureteral  specimen  or  a 
catheterized  bladder  specimen,  it  should  be  treated  as  follows: 

After  obtaining  the  specimen  either  through  a  sterile  ureteral  or 
sterile  urethral  catheter,  rapidly  centrifugalize  the  urine.  Then 
pour  off  the  supernatant  fluid  and  fill  the  centrifuge  tube  with 
sterile  distilled  water,  shake  to  wash  out  the  urinary  salts  which 


THE    STAINING    OF    BACTERIA    IN    URINE  129 

interfere  with  staining,  and  centrifugalize  again.  This  may  be 
repeated,  rejecting  the  supernatant  fluid.  Spread  the  sediment 
upon  a  clean  glass  slide  by  means  of  a  sterile  pipette  or  platinum 
loop,  allow  to  dry  in  the  air,  and  then  fix  by  passing  through 
the  flame  three  times.  Stain  the  specimen  just  as  we  stain  spu- 
tum for  the  Bacillus  tuberculosis,  i.  e.,  steam  for  three  minutes 
with  carbol-f uchsin ;  then  wash  off  the  excess  stain  with  water  and 
decolorize  and  counterstain  with  Gabbet's  solution.  (Gabbet's  so- 
lution is  made  by  mixing  2  grams  of  methylene  blue  with  100  c.c. 
of  25%  sulphuric  acid.)  Dip  the  slide  but  one  minute  in  this 
solution  and  rapidly  wash  off  with  water,  dry,  and  examine. 

If  acid-fast  organisms  are  present,  it  is  well  to  bear  in  mind 
that  not  only  the  Bacillus  tuberculosis  but  also  the  smegma  bacil- 
lus is  acid-fast.  In  other  words,  microscopic  finding  of  an  acid- 
fast  bacillus  in  urine  is  not  positive  proof  of  tuberculosis.  We 
do  not  believe  that  the  differentiation  may  be  made  by  means  of 
the  microscope  alone,  even  though  some  advise  the  expedient 
of  decolorization  with  alcohol  or  with  acid  for  a  longer  time  (the 
smegma  bacillus  does  not  resist  acid  as  long  as  the  tubercle  bacil- 
lus). Rather  would  we  recommend  in  all  cases  the  use  of  the 
guinea  pig  in  making  the  diagnosis  of  renal  tuberculosis.  This 
test  is  carried  out  by  inoculating  with  the  urinary  sediment,  two 
guinea  pigs  that  are  tuberculosis-free,  as  determined  by  the  tu- 
berculin test, — one  intraperitoneally,  the  other  directly  in  the 
mass  of  inguinal  glands.  They  are  kept  under  observation  for 
three  weeks.  If,  during  this  time,  they  have  not  lost  weight  or 
developed  symptoms,  they  usually  show  no  tuberculosis.  How- 
ever, in  the  event  that  the  guinea  pigs  do  not  die  within  this  time, 
they  should  be  kept  three  weeks  longer  and  then  should  be  anes- 
thetized to  death  and  examined  closely  for  signs  of  tuberculosis. 

In  case  there  is  occasion  to  examine  urinary  sediment  for  sim- 
ple organisms  such  as  staphylococci,  etc.,  we  would  recommend 
the  following  procedure :  Treat  the  sediment  as  before,  wash- 
ing out  the  urinary  salts  with  distilled  and  sterile  water.  Smear 
the  sediment  and  dry  on  slides.  Fix  in  flame  and  stain  for  one 
minute  with  Roux's  blue  which  we  have  found  to  be  the  best 
routine  stain  for  bacteria.  Roux's  blue  is  made  as  follows: 


130  BLOOD   AND   URINE    CHEMISTRY 

Solution  A. 

Violet  dahlia  1  gm. 

Absolute  alcohol  10  gms. 

Distilled  water  q.s.  for  100  gms. 

Solution  B. 

Methyl  green  2  gms. 

Absolute  alcohol  20  gms. 

Distilled  water  q.s.  200  gms. 

Prepare  each  solution  separately  by  rubbing  up  the  dye 
with  the  alcohol  in  a  mortar  and  add  the  water  gradually. 
Let  the  mixture  stand  for  24  hours  in  a  bottle.  Then  mix 
the  two  solutions,  filter  and  store  in  a  well-stoppered  bottle. 

After  staining  with  the  above  one  minute,  wash  in  water,  dry, 
and  examine.  This  makes  a  beautiful  stain  for  ordinary  purposes 
and  in  our  experience  is  better  than  the  much  used  Loeffler  stain. 

In  cases  where  Gram  staining  is  necessary,  for  instance,  in  at- 
tempting to  differentiate  gonococci  from  Gram-positive  organisms, 
we  would  recommend  the  following  modification  of  the  usual  Gram 
method.  This  possesses  the  advantage  of  a  permanent  and  re- 
liable primary  stain,  thereby  being  superior  to  the  aniline-oil- 
gentian-violet  mixture  that  must  be  made  up  fresh  every  time  it 
is  used.  Spread  the  urinary  sediment,  dry  in  the  air  and  fix  in 
the  flame. 

1.  Stain  for  30  to  60  seconds  with  carbol-gentian  violet,  which 
is  made  as  follows: 

Gentian  violet  1  gm. 

Carbolic  acid  crystals  2  gms. 

Absolute  alcohol  10  c.c. 

Distilled  water  100  c.c. 

Eub  up  the  gentian  violet  and  the  alcohol  in  a  glass  mor- 
tar, add  the  carbolic  acid  and  mix;  add  two-thirds  of  the 
water,  stirring  all  the  time;  pour  the  mixture  in  a  bottle, 
then  rinse  out  the  mortar  with  the  rest  of  the  water  and 
add  it  to  the  mixture  in  the  bottle.  Leave  for  24  hours  and 
filter  into  a  clean  glass-stoppered  bottle. 

2.  Blot  up  the  excess  of  stain   (but  do  not  wash),  drop  two 
or  three  large  drops  of  Gram's  solution  of  iodine  (iodine  1  gram., 
potassium  iodide  2  grams,  distilled  water  300  c.c.)  on  the  smear, 
and  allow  it  to  stain  20  to  30  seconds. 


THE   STAINING   OF  BACTERIA  IN   URINE  131 

3.  Wash  in  water  and  dry. 

4.  Pour  absolute  alcohol  over  the  film  a  drop  at  a  time  until 
no  more  violet  stain  comes  away — usually  30  seconds. 

5.  Wash    in  water  quickly. 

6.  Counterstain  for  one  minute  with  an  aqueous  solution  of 
saffranin. 

7.  Wash  in  water,  dry  and  examine.     Gram-positive  organisms 
are  stained  a  deep  violet  and  Gram-negative  organisms  a  delicate 
light  pinkish  or  safranin  color. 


CHAPTER  XXIV. 
DESCRIPTION  OF   THE    COLORIMETER. 

The  methods  of  blood  and  urine  chemistry  already  described  en- 
tail the  use  of  the  instrument  known  as  the  colorimeter.  The 
two  best  known  instruments  are  the  Duboscq  and  the  Hellige. 
We  have  already  referred  to  the  fact  that  the  Hellige  is  the 
instrument  of  choice  for  practical  work,  owing  to  its  comparative 
inexpensiveness  and  also  because  much  smaller  quantities  of  flu- 
ids are  necessary  in  using  it,  thereby  saving  considerable  in 
standard  solutions  which  take  time  to  make  and  are  also  very  ex- 
pensive. Possibly  the  Duboscq  is  an  instrument  of  refinement 
and  therefore  particularly  important  in  research  work,  but  for 
the  practical  laboratory  worker  the  Hellige  suffices.  So  far  as 
other  colorimeters  now  on  the  market  are.  concerned,  the  Kutt- 
ner-Leitzj  Myers,  etc.,  we  are  inclined  to  be  dubious  as  to  their 
usefulness  in  work  of  this  kind.  The  disadvantages  of  the  former 
instrument  are  the  use  of  wedges  or  tubes  containing  permanent 
colors  as  standards.  The  Mecca  of  that  success  that  comes  from 
the  greatest  accuracy  is  in  the  rapid  making  and  mixing  of  the 
standard  solutions  at  the  same  time  and  under  the  same  condi- 
tions as  the  unknown.  Standard  solutions  made  in  this  way  and 
used  in  the  colorimeter  are  necessarily  the  best.  The  standards 
for  sugar,  i.  e.,  picramic  acid,  and  the  standard  for  the  functional 
kidney  test  of  Geraghty  and  Rowntree,  keep  some  months,  but 
they  come  within  the  scope  of  the  above  requirements.  We  would 
therefore  exclude  from  consideration  all  colorimeters  using 
wedges  and  tubes  filled  with  solutions  which  are  not  of  the  same 
chemical  structure  and  composition  as  the  unknown.  So  far  as 
the  colorimeter  of  Myers  is  concerned,  it  is  not  to  be  recommended, 
owing  to  the  rapid  changes  that  take  place  in  the  standard  and 
the  unknown  in  the  rather  time-consuming  process  dilution. 

The  following  description  and  drawings  of  the  Hellige  instru- 
ment are  taken  from  the  treatise  by  Prof.  Autenrieth  and  Prof. 


DESCRIPTION    OF    THE    COLORIMETER 


133 


Koenigsberger,  both  of  Freiburg,  published  by  F.  Hellige  & 
Company. 

This  apparatus  is  available  for  color  measurements  of  every 
kind  and  consists  of  a  wooden  case  the  back  and  front  of  which 
are  in  the  form  of  removable  slides,  as  shown  in  Fig.  54. 

The  front  slide  (F)  is  fitted  on  its  outer  side  with  a  slit  plate, 
which  forms  the  observation  window  and  behind  this  on  the  in- 
ner side  is  a  Helmholtz  Double  Plate  (DP).  The  latter  is  mov- 
able and  is  held  between  two  spring  clips  (KL),  from  which  it  can 


Fig.   54. — Representation   of   Hellige   colorimeter. 

be  readily  released  for  the  purpose  of  cleaning.  The  back  (Sch) 
can  be  moved  up  and  down  in  a  convenient  manner  by  means 
of  the  rack  and  pinion  mechanism  (Z),  seen  on  the  right.  The 
back  plate  has  attached  to  it  the  most  essential  part  of  the  colori- 
meter, which  is  a  hollow  glass  wedge  filled  with  a  standard  solu- 
tion. On  the  left  side  the  plate  is  fitted  with  a  scale  (S)  which 
travels  along  a  pointer  (d).  The  open  middle  portion  of  the 
back  between  the  rack  and  the  scale  is  covered  by  a  ground 


134  BLOOD   AND   URINE    CHEMISTRY 

glass  plate  (M),  which,  is  held  in  position  by  a  catch  (h)  at  the 
top  and  may  thus  be  removed  at  any  time  without  trouble. 

Near  the  top  the  sliding  back  is  fitted  with  a  wedge  holder 
(KH)  and  at  a  corresponding  point  at  the  bottom  of  the  slide  it  is 
fitted  with  a  grooved  wooden  block  (B).  To  adjust  the  wedges 
(K)  in  their  proper  position,  the  set  screw  (a)  which  forms  part 
of  the  wedge  holder  should  in  the  first  instance  be  turned  coun- 
ter-clockwise, and  the  fitting  with  the  bracket  attachment  pressed 
firmly  upwards.  The  sealed  end  of  the  wedge  should  then  be 
passed  through  the  hole  in  the  bracket  attachment  and  the  wedge 


Fig.  55. — Representation  of  Hellige  colorimeter. 

let  down  into  the  fitting  and  the  set  screw  turned  clockwise,  so 
as  to  clamp  the  holder  firmly.  The  wedge  should  always  be  in- 
serted with  its  right  angle  and  the  rectangular  vertical  face  turned 
towards  the  observer. 

The  small  glass  trough  (C)  receives  the  liquid  to  be  tested. 
It  slides  into  the  trough  holder  (TH),  whereby  it  is  attached 
to  the  left  side  of  the  colorimeter.  To  set  the  instrument 
for  taking  a  reading,  the  back  of  the  colorimeter  case  together 
with  the  wedge  should  be  moved  up  or  down  bodily  with  the  aid 
of  the  pinion  (21)  and  the  reading  should  be  taken  when  the  color 


DESCRIPTION   OF   THE   COLORIMETER 


135 


intensity  due  to  the  thickness  of  the  standard  fluid  equals  that 
of  the  solution  being  tested. 

To  read  the  result  the  scale  division  indicated  by  the  pointer 
should  be  noted,  and  the  corresponding  figure  read  on  the  ordi- 
nates  of  the  calibration  curve  of  the  standard  wedge ;  and  from  the 
coordinate  abscissa  the  amount  of  substance  contained  in  a  given 
quantity  of  fluid,  as  noted  in  the  curve  table,  can  be  determined. 

The  wedge  should  always  travel  in  close  proximity  to  the 
trough,  which  is  generally  ensured  without  difficulty  by  applying 


Fig.  56. — Representation  of  Hellige  colorimeter. 

a  gentle  pressure  from  the  side.  There  should  never  be  a  bright 
gap  between  the  two  fields  under  comparison,  which  should  merely 
be  separated  by  a  fine  line.  All  glass  fittings,  such  as  the  double 
plate,  trough,  wedge,  and  ground  glass  plate  should  be  dry  on 
the  outside  and  carefully  freed  from  particles  of  dust. 

To  examine  solutions  which  are  so  faintly  colored  as  barely 
to  exhibit  any  tint  when  viewed  in  the  ordinary  trough,  such  as 
when  determining  very  small  quantities  of  ammonia  with  Ness- 


136 


BLOOD   AND    URINE    CHEMISTRY 


ler's  reagent,  it  is  necessary  to  equip  the  colorimeter  with  a  long 
trough  shown  in  Fig.  55.  The  latter  is  supplied  in  two  forms, 
either  with  a  drop-in  cover  (/,  Fig.  55)  or  a  glass  stopper  (g, 
Fig.  55).  This  trough  is  held  in  position  within  horizontal  slides, 
as  shown  in  Fig.  57.  To  put  it  in,  the  ground  glass  back,  should 
be  removed,  the  wedge  put  in  position,  and  the  back  pushed  into 
the  slide  frame.  The  long  trough  with  its  projecting  back  should 
be  passed  through  the  opening  at  the  back  of  the  colorimeter  into 
the  horizontal  -trough  holder  referred  to.  When  the  long  trough 


Fig.  57. — Representation  of  Hellige  colorimeter. 

is  being  used  the  colorimeter  requires  to  be  fitted  at  the  back  with 
a  light-screening  attachment  closed  at  the  end  by  a  ground-glass 
plate  so  as  to  encase  that  part  of  the  trough  which  projects  from 
the  apparatus. 

For  determining  the  proportion  of  iron  present  in  a  solution, 
the  apparatus  is  supplied  with  a  glass  stoppered  trough,  as  shown 
at  e  in  Fig.  55,  so  as  to  obviate  the  evaporation  of  the  ether  during 
the  observation. 


DESCRIPTION    OF    THE    COLORIMETER 


137 


The  various  troughs  may  be  cleaned  by  rinsing  them  out  with 
a  little  diluted  hydrochloric  acid,  after  which  they  should  be 
rinsed  in  rotation  with  water,  alcohol,  and  ether,  and  finally 
dried. 

For  the  success  of  the  colorimetric  method  it  is  essential  that  all 
solutions  so  tested  should  be  absolutely  clear.  All  traces  of  cloudi- 
ness or,  wliat  is  still  more  objectionable,  any  precipitate  that  may 
be  present,  should  be  removed  by  filtration.  The  presence  of  either 
is  liable  to  falsify  completely  the  adjustment  for  equality  of  color 
intensity. 

To  obtain  a  reliable  reading  it  is  best  to  use  diffused  daylight, 
but  it  should  not  be  too  bright.  The  apparatus  should  be  placed 
over  against  a  well  lighted  background,  such  as  a  white  wall, 


Fig.   58. — Optical  arrangement   of  window  of  colorimeter. 

and  the  eye  should  be  applied  to  it  within  the  distance  of  distinct 
vision,  i.  e.,  nearer  than  ten  inches.  After  a  little  practice  use 
may  be  made  of  artificial  light,  but  in  many  cases  the  turning 
point  in  the  intensities  under  comparison  is  not  so  well  marked  as 
when  diffuse  daylight  is  used. 

To  exclude  any  accidental  light,  which  may  interfere  with  the 
accuracy  of  the  reading,  a  screening  tube  about  six  inches  long 
can  be  supplied,  if  desired,  for  attachment  to  the  observation 
window  on  the  front  slide,  which  can  for  this  purpose  be  fitted 
with  a  brass  socket. 

The  instrument  described  above  is  adapted  for  any  species 
of  analysis  by  the  method  of  color  comparison,  and  may  within 
its  proper  limits  be  described  as  a  universal  instrument,  since  by 


138  BLOOD   AND   URINE   CHEMISTRY 

a  simple  interchange  of  standardized  wedges  it  can  be  rendered 
available  for  any  determination  that  may  present  itself.  It  goes 
without  saying  that  every  species  of  analysis  requires  the  use  of 
a  specially  standardized  wedge. 

Special  sets  of  standardized  wedges  are  supplied  for  various 
purposes;  for  instance,  the  analysis  of  drinking  water,  rare  metals, 
etc.  It  is  especially  important  to  note  that  empty  wedges  with 
glass  stoppers  can  be  supplied,  if  ordered,  so  that  the  calibration 
of  new  standards  for  special  colorimetric  determinations  can  be 
undertaken  by  the  analyst  himself. 


PART  III. 

BLOOD  FINDINGS  AND  THEIR 
INTERPRETATION. 


CHAPTER  XXV. 

BLOOD  SUGAR. 

What  is  the  significance  of  the  finding  of  an  undue  amount  of 
sugar  in  blood  as  compared  to  the  finding  of  an  undue  amount 
of  sugar  in  urine?  The  true  condition  of  the  patient  so  far  as 
carbohydrate  metabolism  is  concerned  may  better  be  seen  by  an 
estimation  of  the  amount  of  blood  sugar  that  he  will  show,  rather 
than  by  the  degree  of  glycosuria.  As  a  result  of  the  data  which 
have  been  obtained  by  following  out  these  microchemical  methods, 
we  know  that  a  hyperglycemia  may  exist  without  any  glycosuria. 
Again  we  have  glycosuria  without  hyperglycemia.  The  appear- 
ance of  sugar  in  the  urine  in  cases  of  diabetes  mellitus,  it  is  as- 
sumed, is  merely  a  matter  of  the  threshold  point,  as  it  were,  hav- 
ing been  passed.  The  threshold  point,  that  is,  the  time  when  the 
sugar  increase  in  the  blood  is  accompanied  by  a  pouring  out  of 
sugar  in  the  urine,  is  a  matter  of  debate.  Hammann  and  Hirsch- 
mann,1  at  the  1916  meeting  of  the  American  Society  for  the  Ad- 
vancement of  Clinical  Investigation,  reported  from  a  study  of  50 
cases  that  if  the  blood  sugar  was  not  above  0.17  per  cent,  sugar 
failed  to  appear  in  the  urine,  but  that  when  it  reached  0.18  per 
cent  or  more,  there  was  a  development  of  glycosuria.  Foster,2  at 
the  same  meeting,  found  the  renal  threshold  of  permeability  to  lie 
between  0.149  and  0.164  per  cent,  basing  his  observations  upon 
studies  made  with  patients  after  undergoing  ether  narcosis. 

From  our  own  experience,  there  seems  to  be  great  difficulty 
in  estimating  what  the  normal  threshold  point  is,  and  it  is  for 


1Hammann   and   Hirschmann:      Joslin    (quoted),   Diabetes  Mellitus,    1916,   p.    74. 
2Foster,  N.B. :  loc.  cit. 


140 


BLOOD   AND   URINE    CHEMISTRY 


this  reason  that  blood  sugar  determinations  are  so  vital.  We 
have  data  which  show  higher  concentration  of  sugar  in  blood 
than  are  noted  by  the  above  investigators,  but  these  patients  did 
not  show  glycosuria.  For  instance,  a  very  interesting  case,  which 
was  studied  by  the  authors,  gave  us  a  figure  considerably  higher 
than  that  heretofore  considered  as  the  threshold  point  of  renal 
permeability  for  sugar.  It  will  be  noted  from  a  study  of  the 
figures  shown  in  the  accompanying  chart  of  the  case  of  Mr.  H., 
that  this  individual,  a  diabetic  for  years,  when  starved  for  several 
days,  easily  became  sugar-free  so  far  as  his  urine  was  concerned, 
but  his  blood  sugar  remained  high,  even  though  no  sugar  was 
present  in  the  urine  (Benedict's  test).  It  can  thus  be  seen  that  a 
rather  high  degree  of  hyperglycemia  may  exist  without  any  gly- 
cosuria. This  individual  believed  that  the  few  days'  starvation 
which  made  him  sugar-free  also  placed  him  in  a  state  of  normal 
carbohydrate  equilibrium.  The  result  of  these  blood-  examina- 
tions, however,  convinced  him  of  the  error  of  his  judgment  in 
this  respect. 


CASE    OF   MR.    H. 


BLOOD 

URINE* 

Date 

Sugar  % 

CO2 

Combining 
Power  of 
Blood  Plasma 

Sugar 

Acetone 

Diacetic 
Acid 

7/10/16 

0.330 

68 

7/14/16 

5%  or 
96  gms. 
in  24  hr. 
specimen 

Trace 

Trace 

7/25/16 

0.315 

85 

2.9%  or 
78  gms. 
in  24  hr. 
specimen 

Neg. 

Neg. 

8/16/16 

0.216 



Neg. 

+ 

+ 

8/19/16 

0.165 

53 

Neg. 

+  +  +  + 

+  +  +  + 

=  Small  amount;   +  +  +  +=  Large  amount. 

A  patient  may  be  truly  diabetic  and  may  have  kidneys  relatively 
impermeable  to  sugar  up  to  a  very  high  point.     Hence,  if  only 


BLOOD  SUGAR  141 

the  urine  were  examined  in  such  a  case,  the  negative  findings  would 
not  by  any  means  justify  us  in  eliminating  the  diagnosis  of  diabetes 
mellitus.  Again,  the  finding  of  abundance  of  sugar  in  the  urine 
alone  does  not  give  us  the  most  intelligent  idea  of  the  condition  of 
the  diabetic  and  the  amount  of  starvation  and  dietetic  treatment 
necessary  to  rid  him  of  his  glycosuria  and  his  hyperglycemia.  Rid- 
ding a  patient  with  diabetes  mellitus  of  glycosuria  does  not  by  any 
means  indicate  that  he  is  in  a  state  of  carbohydrate  tolerance. 
We  must,  if  possible,  reduce  his  blood  sugar  to  some  figure  around 
the  normal  of  0.08  to  0.12  per  cent.  If  we  can  make  him  "sugar- 
free"  so  far  as  the  urine  is  concerned,  together  with  low  blood 
sugar  content,  then  we  have  the  case  in  a  condition  where  we 
can  have  some  hope  of  the  performance  of  ideal  .normal  meta- 
bolism. 

Again,  it  must  be  remembered  that  the  advantage  of  a  blood 
chemical  estimation  of  sugar  can  be  seen  from  a  survey  of  the 
opinions  of  the  authorities  as  to  what  constitutes  the  "normal" 
for  sugar  in  the  urine.  Folin3  states  that  he  could  demonstrate 
the  presence  of  sugar  in  human  urine  in  nearly  every  one  of  the 
hundred  persons  upon  whom  he  tried  out  this  procedure  and  adds, 
"The  amount  of  sugar  present  in  normal  human  urine  is  there- 
fore probably  much  greater  than  is  indicated  by  the  negative 
findings  recorded  on  the  basis  of  the  clinical  qualitative  tests  for 
sugar  in  common  use."  Benedict,4  in  a  personal  communication 
to  Joslin,  on  the  other  hand,  claims  that  his  qualitative  test  per- 
formed according  to  his  later  technic  will  detect  glucose  in  as 
low  a  concentration  as  0.01  to  0.02  per  cent,  provided  the  urine 
is  of  low  dilution.  Joslin5  says  that  these  views  hardly  coincide 
nor  do  they  coincide  with  the  views  of  the  older  investigators 
who  supposed  that  normal  human  urine  contained  as  much  as 
0.5  per  cent.  Joslin  further  states6  that,  "It  seems  quite  im- 
possible to  demarcate  sharply  between  normal  and  pathological 
urines  with  reference  to  the  sugar  output."  It  can  thus  easily 
be  seen  that  the  importance  of  blood  sugar  determinations  can- 
not be  overlooked.  Here  we  have  a  doubtful  status  as  to  what 
constitutes  a  "normal"  amount  of  sugar  in  the  urine;  on  the 


'Folin:     Jour.   Biol.   Chem.,   1915,  vol.   xxii,  p.   327. 

'Benedict:     Joslin    (quoted),   Diabetes   Mellitus,   J.   B.    Lippincott   Company,    1916. 

'Joslin:     loc.  cit. 

6Joslin:     loc.  cit. 


142  BLOOD   AND   URINE    CHEMISTRY 

other  hand  there  does  not  seem  to  be  any  doubt  as  to  what  is 
the  normal  for  blood  sugar;  it  lies  between  0.08  and  0.12  per 
cent;  anything  above  this  would  be  termed  hyperglycemia  and 
to  this  figure  we  would  have  to  turn  in  the  presence  of  a  "  doubt- 
ful glycosuria." 

In  our  discussion  of  the  etiology  of  the  disease  diabetes  and  the 
experimental  data  of  later  years  that  have  thrown  so  much  light 
upon  this  question,  we  must  not  forget  to  note  the  pioneer  work 
in  this  field  that  laid  the  basis  for  our  present  scientific  methods. 
Von  Noorden's  work  on  diabetes,7  even  though  his  theoretical 
foundation  has  been  much  disputed,  did  much  to  intensify  the 
interest  in  its  study.  Von  Mering  and  Minkowski,  as  early  as 
1890,  laid  down  certain  truths  about  this  disease  to  which  the 
later  work  of  Allen  possibly  is  attributable.  Others  who  did 
much  in  this  field  were  Lepine,  Arthaud,  Butte,  Remond,  Hedon, 
Gley,  Thiroloix,  Lancereaux,  in  France;  de  Dominicis,  de  Rinzi, 
Reale,  Gaglio,  Caparelli,  in  Italy:  Aldehoff,  Sandmeyer,  Markuse, 
Weintraub,  Seelig,  in  Germany;  V.  Harley,  in  England;  and 
Schabad,  in  Russia.  The  work  of  Minkowski  on  dogs  seemed  to 
crystallize  all  the  previous  thoughts  and  data  into  a  concrete 
whole.  It  might  be  interesting  to  note  that  the  train  of  symptoms 
which  follows  removal  of  all  or  part  of  the  pancreas  in  dogs  is 
about  as  follows:  polyphagia,  polydipsia,  hyperglycemia,  destruc- 
tion of  albumin,  loss  of  weight,  appearance  of  acetone,  diacetic 
acid,  beta-oxybutyric  acid,  ammonia  in  the  urine,  death  in  coma, 
with,  of  course,  glycosuria  at  first  quite  abundant,  later  dwindling 
down  as  the  source  is  depleted. 

It  might  be  well  at  this  point  to  review  some  of  the  facts  of 
normal  and  abnormal  physiological  chemistry  so  far  as  the  source 
and  destiny  of  sugar  in  the  body  is  concerned,  after  which  we 
can  more  intelligently  survey  the  various  classes  of  conditions 
grouped  as  ' ' glycosurias. "  A  glance  at  the  diagrams  (Figs.  59, 
60,  61)  will  show  how  the  sugar  in  the  body  that  is  derived  princi- 
pally from  the  amount  of  carbohydrates  ingested,  is  utilized  under 
normal  conditions.  These  carbohydrates  are  principally  starches 
and  sugars.  The  evolution  of  carbohydrates  in  the  body  takes 
place  by  the  action  of  intestinal  enzymes,  converting  them  into  the 

'von  Noorden:     Die  Zucherkrankheit,  Berlin,  1912. 


BLOOD   SUGAR 


143 


six  hexoses  or  carbon  sugars  which  find  their  way  as  such  into  the 
portal  vein  and  thence  into  the  liver.  In  the  liver  the  sugar  is 
formed  into  glycogen  and  the  excess  sweeps  out  into  the  blood  stream 
via  the  hepatic  vein  as  sugar.  It  is  only  under  exceptional  con- 
ditions that  the  glycogen  stored  in  the  liver  is  called  upon  for 
more  fuel  (sugar).  Experimentally,  of  course,  it  can  be  shown 
that  this  is  true  by  the  finding  of  much  more  sugar  in  the  portal 
vein  than  in  the  hepatic  vein.  The  liver  function  is  possibly  that 
of  a  screen,  holding  back  a  large  part  of  the  sugar  and  allowing 


Liver 


Systemic  circulation 
Glucose  concentration 
0.03% 

Kidneys 


Portal  vein 

Intestinal  tract 


Fig.   59. — Diagram  illustrating  normal  sugar  metabolism.      (From  Forcheimer:  "Therapeu- 
sis  of  Internal  Diseases.") 


Circulation 

ucose  Concentration 
a/a  %  and  more 


Intestinal  tract 


Fig.  60. — Diagram  illustrating  the  nonutilization  of  sugar  in  diabetes.     (From  Forcheimer: 
"Therapeusis  of  Internal  Diseases.") 


"Therapeusi 

the  minor  part  to  go  on  its  way  peripherally.  Of  course  it  must 
not  be  forgotten  that  this  sugar  in  the  circulation  is  not  always 
immediately  demonstrable,  i.  e.,  it  is  stored  up  in  muscle  as  in 
liver  as  glycogen.  The  liver  is  a  veritable  reservoir  of  glycogen. 
It  is  claimed  that  14  per  cent  of  the  weight  of  the  liver  is  fur- 
nished by  its  glycogen  content.  Von  Noorden  very  aptly  calls 
the  liver  a  "glycogen  reservoir"  and  the  muscles  a  "glycogen  de- 
pot." He  means  by  this  that  while  the  percentage  of  glycogen 
in  liver  and  in  muscle  by  weight  is  possibly  identical,  the  call  for 


144  BLOOD   AND   URINE    CHEMISTRY 

glycogen  or  dextrose  is  first  upon  the  liver  and  secondly  upon 
the  muscles.  Another  consideration  of  this  interesting  fact 
would  be  that  the  union  of  the  glycogen  with  the  liver  cells  is 
not  near  so  firm  as  the  union  of  the  muscle  cells  with  their 
glycogenic  visitor.  There  is  another  source  of  sugar,  namely,  pro- 
tein. This  was  disputed  for  a  long  time  but  now  proof  seems  to 
be  undeniable.  Protein  is  transformed  into  amino-acids  such  as 
glycocoll  alanine,  aspartic,  and  glumatie  acids,  and  these  in  turn 
go  over  into  dextrose.  This  was  originally  proved  by  the  experi- 
mental fact  that  animals  fed  exclusively  upon  protein  and  fat 
store  up  large  amounts  of  glycogen. 

A  very  elaborate  research  on  this  question  can  be  found  in  the 
work  of  Kuelz.8  Von  Mering  and  Minkowski,9  in  their  excellent 
work  on  experimental  diabetes,  rather  clearly  prove  the  deriva- 
tion of  some  of  the  sugar  in  the  urine  from  proteins  of  the  food 


Fig.  61. — Diagram  illustrating  excessive  formation  of  sugar  through  nonretention  of  glyco- 
gen in  the  liver.     (From  Forcheimer:  "Therapeusis  of  Internal  Diseases.") 

and  tissues  and  from  fat.  For  the  first  few  days  after  removal 
of  the  pancreas,  it  appears  probable  that  the  sources  of  the  sugar 
are  proteins  and  fats  of  the  body.  The  most  important  point 
from  the  standpoint  of  the  physiologist,  however,  is  the  constant 
relation  between  the  output  of  nitrogen  and  sugar,  the  so-called 
D  :N  ratio  of  experimental  diabetes.  From  the  D  :N  ratio  it  is  safe 
to  conclude  that  dextrose  is  partially  derived  from  protein. 

A  recent  and  most  important  work  bearing  upon  this  point  of 
the  derivation  of  glucose  from  protein  is  that  of  N.  "W.  Janney,10 
who  states  that  the  serious  objections  open  to  the  data  on  this 

8Kuelz:     Reported  in  Pflueger.,  Arch.   f.  d.   ges.    Physiol.,    1903,   vol.   xcvi,   p.    1. 
"von  Mering  and  Minkowski:     Arch.  f.  d.  ges.   Physiol.,  1904,  vol.   cvi,  p.    160. 
lujanney,  N.  W.:  Arch.  Int.   Med.,  Nov.   15,   1916,  vol.  xviii,  No.   5,  p.  584. 


BLOOD   SUGAR  145 

line  of  work  in  the  past  are  based  upon  the  fact  that  the  feeding 
experiments  are  not  conclusive,  inasmuch  as  it  cannot  be  demon- 
strated that  all  the  food  material  is  digested  and  absorbed  and 
that  all  the  glucose  arising  from  this  material,  and  no  more, 
originates  from  the  protein  that  has  been  given  the  subject.  It 
must  be  remembered,  too,  that  in  diabetes  mellitus  a  certain  amount 
of  oxidation  takes  place  and  that  the  capacity  of  the  average 
human  diabetic  to  utilize  glucose  frequently  may  undergo  con- 
siderable daily  variation,  even  when  the  diet  remains  the  same. 
It  is  also  possible,  states  Janney,  that  the  glucose  originating 
from  food  protein  may  be  in  part  synthetically  used  in  the  for- 
mation of  various  body  substances  or  may  be  deposited  as  glyco- 
gen.  Again  it  is  inadvisable  to  use  fasting  diabetics  for  these  ex- 
periments because  starvation  increases  the  ability  of  the  organ- 
ism to  oxidize  glucose.  Another  and  contrary  effect  of  feeding 
quantities  of  sugar-forming  proteins  to  diabetics  is  to  lower  the 
tolerance  of  the  organism  for  glucose.  This  is  very  evident  from 
data  accumulated  experimentally  by  Mohr.  Another  disturbing 
factor  in  using  the  human  diabetic  is  the  fact  that  muscular  ex- 
ercise may  decrease  the  glycosuria  under  some  circumstances  and 
increase  it  under  others.11  The  difficulty  of  preventing  diabetics 
from  breaking  diet  is  the  chief  cause  of  the  error  in  human  ex- 
periments. Using  dogs  with  extirpation  of  the  pancreas  has  been 
attempted,  in  these  experiments,  but  this  is  a  poor  method  be- 
cause extirpation  of  the  pancreas  in  dogs  is  followed  by  severe 
affections  of  the  digestive  system. 

With  these  facts  in  mind,  Janney  tried  out  these  experiments 
in  the  course  of  cases  of  phlorizin  diabetes,  developing  a  technic 
by  which  the  extent  of  protein  conversion  into  glucose  could  be 
followed  with  considerable  accuracy.  The  details  of  this  technic 
may  be  found  in  his  previous  publications.12  Janney  mentions  a 
few  facts  about  phlorizin  diabetes  which  has  been  so  well  studied 
of  late  years  by  Lusk  and  others  (see  page  150  for  further  particu- 
lars on  phlorizin).  Where  phlorizin  is  given  to  dogs,  diabetes  de- 
velops, the  reserve  of  carbohydrates  in  the  body  is  used  up,  and  in 
the  fasting  state  the  glucose  appearing  in  the  urine  bears  a  con- 


llvon   Noorden:      Die  Zuckerkrankheit,    1912,   ed.   6,   p.    100. 
"Janney,  N.   W. :     Jour.  Biol.   Chem.,  1915,  vol.  xx,  p.   321. 

Janney,  N.   W.,  and  Csonka,  F.  A.:  ibid.,  vol.  xxii,  p.   203. 

Janney,  N.   W.,  and   Blatherwick,   N.   R. :   ibid.,  vol.   xxiii,  p.  77. 


146  BLOOD   AND   URINE    CHEMISTRY 

stant  relation  to  the  urinary  nitrogen,  this  so-called  glucose- 
nitrogen  ratio  averaging  3.4  to  1.  Glucose  administered  to  such 
dogs  is  quantitatively  excreted.13  Glucose  arising  from  nontoxic 
ingested  substances  fails  to  be  stored  up  but  appears  in  the  urine 
as  such.  Janney's  experimental  work  shows  that  it  is  probable 
that  all  the  glucose  arising  from  protein  fed  to  phlorizined  dogs  is 
excreted  in  their  urine.  This  demonstrates  that  the  urinary  glu- 
cose and  nitrogen  of  fasting  phlorizined  dogs,  which  quantita- 
tively excrete  ingested  sugar,  bear  the  same  relation  to  each  other 
as  the  extra  glucose  arising  from  these  animals'  own  protein  in- 
gested by  other  phlorizined  dogs  does  to  the  nitrogen  contained  in 
these  proteins.  The  sugar  excreted  under  these  circumstances 
represents  the  maximal  amount  formed  from  the  animals'  body 
proteins. 

Janney's  work  showed  that  glucose  formation  from  protein  is 
the  same  in  diabetes  mellitus  as  in  phlorizin  diabetes.  He  found 
that  isolated  proteins  yielded  large  amounts  of  glucose  in  metabol- 
ism, varying  from  48  to  80  per  cent  according  to  the  protein  ex- 
amined. Contrary  to  existing  opinions,  the  animal  or  vegetable 
origin  of  proteins  bears  no  relationship  to  their  ability  to  produce 
glucose  in  the  animal  organism,  this  function  being  found  to  be 
mainly  dependent  on  the  amounts  of  sugar-yielding  amino-acids 
entering  into  the  constitution  of  these  various  proteins.  Janney's 
studies  on  glucose  formation  from  body  proteins  demonstrate  that 
body  proteins  of  man  and  animals  yield  about  58  per  cent  of  glu- 
cose in  metabolism.  The  nitrogen  of  these  proteins  bears  about 
the  relation  of  3.6  to  1  to  the  glucose  formed  from  them.  This 
definite  establishment  of  the  glucose-nitrogen  (D:N)  ratio  is  of 
value  in  the  prognosis  of  diabetes.  Cases  showing  a  high  urinary 
D  :N  ratio  averaging  3.4  to  1,  are  to  be  regarded  as  grave.  The 
lower  the  ratio,  the  more  favorable  the  prognosis.  As  the  glucose 
eliminated  by  the  fasting  diabetic  is  of  protein  origin,  sugar  forma- 
tion from  fat  does  not  take  place  to  any  great  extent  in  this  dis- 
ease. 

Janney  also  reported  the  results  of  glucose  formation  from  pro- 
tein foods,  using  the  same  technic.  In  von  Noorden's  food  tables 
for  diabetics,  glucose  formation  from  protein  has  not  been  taken 

"Kinger,  A.  I.:     Jour.  Biol.   Chem.,  1912,  vol.  xxii,  p.   422. 


BLOOD   SUGAR  147 

into  account.  By  adding  the  amounts  of  glucose  yielded  in  meta- 
bolism by  the  proteins  of  a  given  food  to  its  carbohydrate  content, 
it  is  possible  to  ascertain  the  actual  amount  of  sugar  both  set  free 
and  formed  in  the  metabolism  of  such  foods.  Janney  also  found 
from  experimental  studies  that  the  various  proprietary  protein 
foods  present  no  advantages  over  equal  amounts  of  bread  when 
fed  to  diabetics,  as  the  large  amount  of  protein  present  leads  to 
the  formation  of  considerable  glucose  in  metabolism. 

When  we  come  to  the  consideration  of  the  possibility  of  the 
derivation  of  dextrose  in  the  body  from  fat,  we  have  not  yet  had 
sufficient  experimental  or  chemical  proof.  We  know  that  in  plant 
life  carbohydrates  seem  to  undergo  transformation  into  fat,  still 
it  has  not  yet  been  clearly  proved  in  the  animal  economy.  Foster, 
in  his  excellent  work,14  calls  attention  to  this  point,  quoting  from 
analyses  of  nuts  by  du  Sablon.15  The  figures  are  parts  per  100. 

OIL  GLUCOSE 

On  July  6,  these  nuts  showed     3  7.6 

Aug.  1,      "       "         "16  2.4 

Sept.  1,      "       "         "       59  0 

Oct.    4,      "       "         "       62  0 

Again  we  have  the  example  of  the  germination  of  seeds  with  the 
disappearance  of  fats  and  the  appearance  of  carbohydrates.  These 
facts  of  plant  physiological  chemistry  do  not  hold  good,  however, 
with  the  animal  organism.  Fats  are  split  up  into  glycerol  and  the 
fatty  acids,  but  so  far  there  is  no  proof  of  their  ultimate  conversion 
into  sugar.  We  know  now  that  the  increase  of  fats  in  the  diet  of  a 
diabetic  does  not  increase  the  amount  of  sugar  in  the  urine.  The 
von  Noorden  idea  on  diabetes  has  been  shown  to  be  erroneous,  par- 
ticularly with  reference  to  the  fact  that  sugar  in  any  quantity  re- 
sults from  the  catabolism  of  fat. 

The  ultimate  fate  of  dextrose  in  the  body  is  not  clearly  and 
definitely  understood.  While  we  have  many  theories  and  many 
experiments,  we  cannot  place  our  finger  firmly  and  definitely  upon 
the  pivotal  point  of  the  change  of  a  normal  person,  say,  into  a 
diabetic.  As  Foster16  truly  says:  "At  the  present  time  we  must 
confess  that  we  are  quite  without  sufficient  data  to  form  any  clear 
conception  of  the  breakdown  of  the  glucose  molecule,  and  it  is  prob- 

"Foster,  N.  B.:     Diabetes  Mellitus,  J.  B.   Lippincott  Company,  1915. 

15du  Sablon:     Compt.   rend.,   1896,  vol.   cxxiii,  p.   1084. 

"Foster,  N.B:     Diabetes  Mellitus,  J.    B.  Lippincott   Company,   1915. 


148  BLOOD   AND   URINE    CHEMISTRY 

able  in  the  initial  step  in  the  destruction  of  glucose  that  the  es- 
sential deviation  of  the  diabetic  from  the  normal  becomes  manifest. 
Certainly  the  diabetic  organism  is  usually  able  to  handle  the  cleav- 
age products  of  glucose.  The  inability  to  effect  the  first  cleavage 
might  rest  in  a  change  in  the  cell  where  oxidation  is  effected  or  in 
the  absence  of  an  activator.  In  the  light  of  our  knowledge  of  other 
vital  processes,  we  must  assume  the  dependence  of  these  changes 
upon  zymases  elaborated  in  one  class  of  cells,  perhaps  the  muscle, 
and  in  order  to  effect  their  function  probably  requiring  an  acti- 
vator or  hormone  secreted  perhaps  by  quite  remote  and  different 
cells. 

Joslin17  states  that  he  considers  every  patient  a  diabetic  un- 
til the  contrary  is  proved,  who  has  sugar  in  his  urine  demon- 
strable by  any  of  the  common  tests.  At  this  point  it  must  be 
remembered  that  glycosuria  simply  means  sugar  in  the  urine  in 
undue  quantities.  How  this  may  be  brought  about  independent 
of  the  disease  diabetes  mellitus,  we  shall  now  consider.  Every 
medical  man  is  familiar  with  the  classic  experiment  of  Claude 
Bernard,18  who,  as  early  as  1845,  induced  glycosuria  in  rabbits 
by  his  piqure  experiment,  i.  e.,  the  insertion  of  a  steel  stylet  into 
the  brain  of  a  rabbit.  Bernard  thrust  his  stylet  into  the  inferior 
part  of  the  calamus  scriptorius.  This  glycosuria  persisted  sev- 
eral hours  provided  the  animals  were  in  a  normal  state  of  nutri- 
tion. It  was  completely  inhibited  if  the  animal  had  fasted  for  a 
period  prior  to  the  experiment,  in  other  words,  if  its  glycogen 
had  been  practically  released  and  burned  up  from  its  "reser- 
voir" in  the  liver.  The  blood  sugar,  as  well  as  urine  sugar,  rises 
in  puncture  diabetes.  Bernard  also  showed  that  nerve  stimula- 
tion had  a  profound  influence  in  these  experiments.  The  stimula- 
tion of  the  splanchnics  by  the  stylet  in  the  so-called  "diabetic 
center,"  of  course,  causes  the  liberation  of  the  glycogen  in  liver 
and  its  undue  appearance  in  blood,  thence  into  urine.  Stimula- 
tion of  the  cut  vagi  after  puncture  of  the  calamus  scriptorius 
caused  the  following :  stimulation  of  the  central  stump  induced  gly- 
cosuria; stimulation  of.  the  peripheral  stump  did  not.  Eckhard19 
showed  that  division  of  the  vagus  and  electrical  stimulation  will 

17Joslin:  loc.  cit. 

"Bernard,    Claude:     De  1'origine   du  sucre   dans  1'economie   animale,   Paris,    1848;  -also 
Lecons  sur  le  diabete  et  la  glycognese  animale,  J.   B.   Balliere  et  fiis,   1877,  p.   576. 
"Eckhard:     Beitr.  z.  Anat.  u.  Physiol.,  1896,  vol.  iv,  p.  4. 


BLOOD   SUGAR  149 

cause  temporary  glycosuria  even  several  days  after  the  nerve  is 
divided.  Sugar  may  also  be  caused  to  appear  in  the  urine  by 
cutting  the  lower  cervical  or  upper  thoracic  sympathetic  ganglia, 
as  shown  by  Sehiff.20 

It  is  also  noteworthy  that  the  adrenal  bodies  are  somehow  con- 
cerned in  glycogenolysis  and  glycosuria.  It  was  Herter21  who 
first  showed  that  painting  the  pancreas  with  adrenal  extract 
caused  glycosuria.  The  application  of  adrenal  extracts  has  a 
profound  influence  upon  hyperglycemia  and  glycosuria.  We 
have  alluded  before  to  the  fact  that  the  liver  combination  with 
glycogen  is  not  nearly  so  firm  as  the  muscle  combination,  yet 
the  injection  of  epinephrin  into  the  blood  causes  the  liberation 
of  sugar  more  quickly  from  the  muscles  than  from  the  liver,  ac- 
cording to  Kutschmer.22  When  animals  are  made  glycogen-free 
by  fasting  and  the  use  of  phlorizin,  the  use  of  epinephrin  does 
not  produce  glycosuria,  indicating  that  this  too,  like  the  piqure 
of  Bernard,  is  a  form  of  glycogenolysis.  It  is  a  fact  that  piqure 
glycosuria  does  not  occur  if  the  adrenals  are  previously  removed, 
indicating  the  influence  of  these  bodies  upon  this  experiment. 
Other  agents,  that,  like  epinephrin,  act  upon  the  peripheral  blood 
vessels  and  cause  vasoconstriction  with  raising  of  blood  pressure, 
produce  glycosuria  ;  for  instance,  barium  chloride.  This  was  shown 
by  Neubauer.23  He  showed  also  that  drugs  which  cause  vaso- 
dilatation  prevent  glycosuria  due  to  epinephrin,  e.g.,  pilocarpine 
and  nicotine. 

It  might  be  well  to  mention  the  fact  that  not  only  actual  punc- 
ture of  the  calamus  scriptorius  causes  glycosuria  transitoria  ;  in- 
crease in  intracranial  pressure  or  traumatic  pathological  condi- 
tions of  other  kinds  may  do  so.  One  of  us24  reported  an  observa- 
tion of  severe  and  transitory  glycosuria  in  a  case  of  cerebral 
hemorrhage  due  to  an  intraventricular  hemorrhage.  In  this  case 
the  glycosuria  lasted  several  days  and  disappeared,  possibly  co- 
incidently  with  the  using  up  of  all  the  glycogen  in  the  liver  and 
muscles.  Autopsy  later  showed  the  clot. 


20Schiff:      Unterschung    fiber    die    Zuckerbilding    in    der    Leber    u.    den    Einfluss    des 
Nervensystems  auf  die   Erzeugung  des  Diabetes,   Wvirzburg,    1859. 
"Herter:     Medical  News,   1902. 

22Kutschmer:      Arch.    f.   exper.   Path.   u.    Pharmakol.,    1907. 
^Neubauer:     Biochem.  Ztschr.,   1912,  vol.  xliii. 
^Gradwohl,  R.   B.  H.:  Philadelphia  Med.  Jour.,  April  22,   1839. 


150  BLOOD   AND   URINE    CHEMISTRY 

It  is  claimed  by  Woodyatt25  that  various  other  drugs,  such  as 
phosphorus,  carbon  monoxide,  hydrazine,  arsenic,  etc.,  may  cause 
glycosuria  by  causing  the  increased  glycogenolysis  alluded  to 
above. 

Another  interesting  form  of  glycosuria  is  that  caused  by  the 
injection  of  phlorizin,  called  "phlorizin  diabetes."  Eeferences 
to  this  interesting  condition  can  be  found  in  the  literature.26 

Phlorizin  is  a  glucoside  which  can  be  extracted  from  the  bark 
of  apple  and  cherry  trees.  In  1886,  von  Mering  established  the 
fact  that  the  administration  of  this  drug  to  dogs,  geese,  and  rab- 
bits induced  glycosuria.  If  you  give  a  dog  1  gm.  of  phlorizin 
per  kilo  of  body  weight,  in  a  few  hours  you  will  observe  at  least 
10  per  cent  of  urine  sugar.  The  blood  sugar  will  not  rise.  In 
other  forms  of  diabetes  except  this  variety,  you  have  hyperglycemia. 
The  sugar  will  persist  in  the  urine  as  long  as  you  give  the  phlorizin. 
All  sugar  as  it  is  formed  in  the  body  goes  out  in  the  urine  as 
sugar.  It  is  claimed  by  some  that  in  this  condition  the  phlorizin 
ingested  simply  throws  down  the  barrier  of  the  kidney  filter;  in 
other  words,  that  the  kidneys  are  made  absolutely  and  completely 
permeable  to  sugar  by  some  alteration  in  the  secretory  epithelia. 
This  overflow  of  sugar  from  the  blood  causes  a  deficit  which  is 
supplied  by  the  pouring  out  of  more  glycogen  from  liver  and 
muscles  into  the  circulating  blood  as  sugar,  until  all  is  used  up. 
It  is  for  this  reason  that  there  is  no  undue  accumulation  of  sugar 
in  the  blood.  Here  again  we  wish  to  allude  to  von  Noorden's 
ideas,  that  at  this  juncture  he  thought  the  supply  of  sugar  in 
phlorizin  diabetes  was  replenished  by  protein  and  fatty  tissues 
of  the  body. 


^Woodyatt,   R.    T.:    quoted   in:    Wells'    Chemical    Pathology,    Philadelphia   and   London 
second  edition,  1914,  p.  573. 

26von    Mering:      Cong.    f.    inn.    Med.,    1886,   vol.    clxxxv;    Ztschr.    f.    klin.    Med.,    1889, 
vols.  xiv  and  xvi. 

Moritz  and  Prausnitz:  Ztschr.  f.   Biol.,   1891,  vol.  xxvii. 

Kuelz  and  Wright:     Ztschr.  f.  Biol.,  1890,  vol.  xxvii. 

Cremer  and  Ritter:     Ztschr.  f.   Biol.,   1892,  vol.  xxviii. 

Minkowski:      Arch.    f.    exper.   Path.    u.    Pharmakol.,    1893,   vol.    xxxi. 

Zuntz:     Verhandl.  d.   physiol.  Gesellsch.,  Berlin,   1895,  5,  vol.  vii. 

Levene:     Jour.  Physiol.,  1894,  vol.  xvii,  p.  259. 

Coolen:     Centralb.  f.  d.  Krankh.  d.  Ham-  Sex.-Org.,   1895,  vol.  vi,  p.   530 

Pavy:     Jour.  Physiol.,   1896,  vol.  xx. 

Contejean:     Compt.   rend.    Soc.   de  biol.,   1896,  vol.  xlviii,   p.   344. 

Markuse:     Allg.  med.   Centr.-Ztg.,   1896,  No.   49. 

Klemperer:     Verhandl.  d.  Ver.   f.  inn.   Med.,   1896,  vol.  v,  p.   18. 

Lepme:      Semaine  med.,   1895,  p.   383. 

Kolish:     Wien.    klin.    Wchnschr.,    1897,   No.   23. 

Lusk,  G.:     Ztschr.  f.  Biol.,  1898,  vol.  xxxvi,  p.   82. 


BLOOD   SUGAR  151 

It  is  curious  that  in  a  very  modern  and  recent  publication,27 
a  writer  calls  attention  to  the  fact  that  phlorizin  glycosuria  is 
sometimes  called  "renal  diabetes"  (italics  ours)  just  as  some  of 
the  older  writers  spoke  of  a  condition  which  we  shall  presently 
take  up,  namely,  "renal  diabetes."  But  it  is  our  impression  that 
phlorizin  diabetes  and  renal  diabetes  are  in  no  way  related  and 
should  not  be  confused.  It  is  true  that  in  both  conditions  there 
is  glycosuria  but  no  hyperglycemia,  but  otherwise  there  is  no 
pathology  known  for  either.  We  must  sharply  distinguish  ' '  renal 
diabetes"  from  diabetes  mellitus,  although  we  have  but  little 
pathology  on  which  to  base  the  gross  or  minute  differentiation. 
Foster28  and  Joslin,29  who  have  written  the  most  recent  works  on 
diabetes  mellitus,  both  insist  that  the  future  conception  of  this 
so-called  "renal  diabetic"  state  must  rest  upon  blood  chemical 
analyses.  Foster  offers  the  suggestion  that  these  cases  of  renal 
diabetes  are  really  cases  of  beginning  diabetes  mellitus,  but  we 
must  confess  that  the  blood  data  on  such  cases  does  not  justify 
this  classification.  Our  conception  of  a  true  case  of  diabetes  mel- 
litus is  one  with  definite  hyperglycemia  and  with  possibly  gly- 
cosuria. If,  therefore,  we  meet  with  a  case  that  shows  no  hyper- 
glycemia and  with  definite  increase  over  the  normal  values  of  the 
urine  sugar,  we  must  classify  this  until  further  notice  as  a  case 
of  renal  diabetes.  The  cases  of  renal  diabetes  so-called  that  occur 
during  the  pregnancy  period  are  sufficiently  illuminating  to  bear 
description.  It  is  well  known  that  in  the  pregnant  state  sugar 
may  at  times  be  found  in  the  urine  but  no  increase  of  blood  sugar 
occurs;  besides,  there  are  no  signs  or  symptoms  of  diabetes 
mellitus  and  the  occurrence  and  presence  of  the  sugar  in  the  urine 
in  no  way  seems  to  influence  for  the  bad  the  pregnant  status. 
These  women  after  the  puerperium,  show  no  glycosuria,  and 
yet  when  they  become  pregnant  again,  again  show  glycosuria. 
They  are  justly  entitled  to  be  called  renal  diabetics  and  in  no 
sense  "incipient"  cases  of  diabetes  mellitus. 

In  passing,  we  therefore  urge  the  use  of  blood  analytical  chemi- 
cal methods  in  seeking  more  light  upon  the  differential  diag- 
nosis of  renal  diabetes  and  diabetes  mellitus.  Joslin,  who  has 


"Monographic  Medicine,  D.  Appleton  &  Co.,  N.  Y.,  1916,  vol.  Hi,  p.  788. 
^Foster:     loc.  cit. 
^Joslin:     loc.  cit. 


152  BLOOD   AND   URINE    CHEMISTRY 

had  a  very  wide  experience  in  handling  and  studying  diabetes 
mellitus,  states  that  "renal  diabetes  rarely  occurs.  The  re- 
sults of  the  demonstration  of  the  percentage  of  sugar  in  the 
blood  of  diabetics,  which  are  now  being  rapidly  accumulated  will 
throw  light  upon  this  question.  Seven  cases  of  my  series  must 
be  more  carefully  studied  with  this  in  mind.  As  yet  I  am  not  in- 
clined to  classify  any  of  these  as  renal  diabetes."  In  examining 
the  discussion  alluded  to  by  Joslin  we  regret  to  note  that  his 
observation  of  the  blood  sugar  did  not  occur  on  the  same  day  as 
his  observation  of  the  urine  sugar;  manifestly  giving  us  no 
basis  for  watching  the  ratio  of  subsidence.  He  states  that  ''the 
urine  was  usually  sugar-free  at  both  the  time  of  the  first  and 
last  blood  tests.  It  will  be  of  interest  to  compare  these  figures 
with  those  observed  with  a  subsequent  series  of  patients.  It  seems 
remarkable  that  so  many  patients  should  become  sugar-free  and 
yet  the  blood  sugar  remain  so  high.  Presumably  this  is  due  to 
the  short  period  of  time  intervening  between  the  first  and  the 
last  blood  test.  It  would  seem  to  indicate  that  rigorous  dietetic 
treatment  should  be  continued  even  for  a  long  period  of  time 
after  the  patient  becomes  sugar-free." 

A  very  interesting  contribution  to  the  literature  of  renal 
diabetes  is  a  recent  article  by  Lewis  and  Mosenthal.30  They  state 
that  in  this  condition  the  blood  sugar  does  not  vary  from  the 
bounds  of  the  normal,  an  increase  or  decrease  in  the  carbohydrate 
diet  has  little  effect  on  the  percentage  of  sugar  in  the  blood  or  the 
quantity  excreted  in  the  urine.  These  cases  have  none  of  the 
clinical  manifestions  of  diabetes  mellitus,  due  either  to  diminished 
ability  of  the  body  to  utilize  glucose  or  the  presence  of  a  hyper- 
glycemia;  there  is  no  polydipsia,  polyphagia,  or  polyuria,  no 
loss  of  weight  or  weakness,  no  pruritus  or  furunculosis,  nor  any 
other  symptom  of  this  disease.  It  remains  stationary,  the  gly- 
cosuria  shows  no  tendency  to  increase,  nor  does  diabetes  mellitus 
develop  from  it ;  the  subject  continues  in  good  health  and  without 
any  abnormal  symptoms  except  a  constant  low  grade  glycosuria. 
The  data  necessary  for  the  diagnosis  of  renal  diabetes  are  very 
few  in  number,  but  sharply  defined : 

1.  A  glycosuria,    maintained    at    a    fairly    constant    level    and 

30Uwis  and  Mosenthal:     Bull.  Johns  Hopkins  Hosp.,  1916,  vol.   xxvii,  No.   303,  p.   133. 


BLOOD   SUGAR  153 

not  markedly  affected  by  changes  in  the  carbohydrate  content  of 
the  food. 

2.  A  normal  percentage  of  blood  sugar  while  the  urine  con- 
tains glucose. 

Cases  in  the  literature  are  not  very  common.  Von  Noorden 
was  somewhat  skeptical,  but  Allen31  admits  two  cases,  those  of 
Bonniger  and  Taehau,  as  absolute  examples  of  the  condition. 
Other  cases  are  those  by  Graham32  and  de  Langen.33  Lewis  and 
Mosenthal's  case  report  is  another  undoubted  case  added  to  the 
literature.  The  full  history  of  this  interesting  case  is  as  follows : 

W.  P.  W.,  Medical  History  No.  34774,  male,  white,  age  29,  born  in  the  U.  S., 
a  station  agent,  descended  from  Anglo-Saxon  ancestry. 

Family  History. — Father  (aged  60),  mother  (aged  50),  one  brother  and  four 
sisters  are  all  alive  and  in  good  health ;  one  sister  died  of  erysipelas.  With  the 
exception  of  marked  obesity  in  one  grandmother  and  several  of  her  sisters,  there 
is  no  history  of  hereditary  disease ;  diabetes  mellitus,  heart  trouble,  kidney  dis- 
ease, apoplexy,  gout,  exophthalmic  goiter,  and  tuberculosis  have  never  been 
found  in  the  patient 's  family. 

Habits. — Smokes  five  to  six  pipes  a  day ;  does  not  use  alcohol ;  eats  a  con- 
siderable amount  of  bread  but  no  excess  of  sweets. 

Past  History. — Measles  and  whooping  cough  in  childhood,  malaria  18  years 
ago,  pneumonia  17  years  ago,  varicella,  complicated  by  otitis  media  on  the 
right  side,  15  years  ago.  Venereal  infection  is  denied. 

Present  History. — Three  years  ago  passed  a  life  insurance  examination. 
This  is  the  only  urinary  test  remembered,  until  six  weeks  ago,  when  the 
patient  applied  to  his  physician  for  relief  from  backache.  At  that  time  a 
glycosuria  was  demonstrated.  The  backache  cleared  up  shortly;  the  glyco- 
suria persisted  in  spite  of  a  restriction  of  the  carbohydrates  in  food.  There 
never  have  been  any  other  symptoms  pointing  to  diabetes  mellitus  with  the 
exception  of  transient  paresthesia  of  the  fingers  (no  loss  of  weight  or 
strength,  no  polyuria,  polyphagia,  no  skin  involvement — pruritus,  furunculosis 
or  other  condition — no  muscular  cramps,  no  pains  in  the  extremities)  ;  there 
have  been  no  evidences  of  pancreatic  disease  (no  pain  in  the  epigastrium,  no 
fatty  diarrhea) ;  all  indications  of  exophthalmic  goiter  have  been  completely 
lacking  at  all  times  (no  exophthalmos,  no  thyroid  enlargement,  no  vomiting, 
nervousness,  cardiac  palpitation,  or  diarrhea) ;  there  have  been  no  signs  of 
acromegaly  or  giantism  pointing  to  a  hypophyseal  involvement;  there  has 
been  no  history  of  a  renal  lesion  (no  headache,  visual  disturbance,  dyspnea, 
vertigo,  edema  or  albuminuria) ;  there  has  never  been  any  skin  pigmentation 
to  suggest  a  cirrhosis  of  the  pancreas  and  liver,  that  is  hemachromatosis. 

For  the  last  two  or  three  years  there  has  been  a  tendency  to  increased 
frequency  of  urination  during  the  day  but  not  at  night.  The  quantities 
voided  have  apparently  not  exceeded  normal.  This  is  evidently  a  pollakiuria 

"Allen:     Glycosuria  and   Diabetes,   Boston,   1913. 

32Graham:     Jour.    Physiol.,    1915,  vol.  xlix,   p.   46    (proceedings). 

33de   Langen:      Berl.   klin.    Wchnschr.,    1914,   vol.   li,   p.    1792. 


154  BLOOD   AND   URINE    CHEMISTRY 

rather  than  a  polyuria,  which  is  borne  out  by  the  ward  observations  which 
will  be  detailed  further  on. 

There  has  been  a  slight  chronic  cough  associated  with  a  moderate  nasal 
catarrh,  and  mouth  breathing.  There  have  been  no  night  sweats,  hemoptysis, 
or  "pleuritic  pain." 

Present  Complaint. — The  patient  feels  perfectly  well  and  would  not  be- 
lieve himself  sick  were  it  not  for  the  persistent,  "  sugar-in- the-urine. " 

Physical  Examination. — Height  5  feet,  9%  inches,  weight  152  pounds;  ap- 
pears to  be  in  the  best  of  health  and  spirits;  the  skin  and  mucous  membranes 
are  not  pigmented,  their  color  is  normal,  they  are  as  moist  as  those  of  a 
normal  individual.  The  pupils  are  equal  and  react  to  light  and  accommoda- 
tion; Von  Graefe's  sign  is  absent.  The  pharynx  is  injected  and  there  is  a 
moderate  degree  of  nasal  obstruction,  as  indicated  by  persistent  mouth 
breathing.  The  tonsils  are  not  enlarged  or  inflamed.  There  is  no  pyorrhea 
alveolaris.  The  thyroid  is  barely  palpable.  The  pulse  rate  averages  75;  the 
pulse  is  regular  in  force  and  frequency  and  of  normal  value.  The  radial 
artery  can  be  rolled  under  the  palpating  finger,  but  is  soft  and  elastic.  The 
temperature  is  normal.  The  respiratory  rate  ranges  from  16  to  24.  The 
systolic  blood  pressure  is  140,  the  diastolic  80.  The  heart 's  apex  beat  cannot 
be  seen,  it  is  barely  palpable  in  the  fifth  interspace,  10  cm.  to  the  left  of  the 
median  line;  the  character  of  the  apex  impulse  is  a  normal  one;  there  are  no 
thrills  over  the  precordium;  the  area  of  relative  cardiac  dullness  extends  3.5 
cm.  to  the  right  of  the  mid-line  in  the  fourth  space,  and  10.5  cm.  to  the  left 
in  the  fifth;  the  heart  sounds  reveal  no  murmurs,  the  second  sound  over  the 
aortic  area  is  somewhat  intensified  and  is  louder  than  the  pulmonic  second 
sound.  The  lungs  are  normal  except  for  slight  dullness  and  somewhat  pro- 
longed expiration  in  the  right  supraspinous  fossa,  and  at  times  a  few  dry 
rales,  after  coughing,  over  the  same  area.  The  liver  and  spleen  are  not  pal- 
pable and  there  are  no  areas  of  tenderness  or  increased  resistance  over  the 
abdomen.  The  patellar  reflexes  are  very  active.  There  is  no  edema  of  the  face, 
back  or  extremities.  On  the  left  thigh  there  is  a  small  eczematous  patch  fur- 
rowed by  scratch  marks.  The  superficial  lymph  nodes  are  not  enlarged.  The 
hemoglobin  is  100  per  cent  (Sahli),  the  red  blood  cells  are  4,000,000  and  the 
white  blood  cells  are  8450  per  c.mm.  The  Wassermann  test  is  negative. 
The  urine  on  admission  is  clear,  of  reddish  yellow  color,  specific  gravity  1035, 
acid  in  reaction,  negative  for  albumin,  gives  a  distinct  reaction  for  sugar, 
and  on  microscopic  examination  yields  no  casts  or  red  blood  cells;  the  quali- 
tative tests  for  acetone  and  diacetic  acid  are  negative;  the  'phthalein  test 
shows  an-  excretion  of  42  per  cent  in  two  hours;  Ambard's  constant  deter- 
mined at  various  times  is  0.07,  0.11,  0.08,  0.10. 

Impression. — The  presence  of  glycosuria  was  well  established.  The  urine 
gave  a  positive  reaction  with  both  the  quantitative  and  qualitative  Fehling- 
Benedict  reagent,  yielded  gas  on  fermentation  with  yeast,  and  the  unfer- 
mented  urine  rotated  the  polariscope  to  the  right.  The  nature  of  the  gly- 
cosuria will  be  subsequently  discussed.  There  may  have  been  a  healed  tuber- 
cular lesion  at  the  right  apex;  impaired  resonance,  slightly  prolonged  expira- 
tion and  inconstant  rales  in  this  region  are  not  pathognomonic  of  a  tubercu- 
lous focus;  it  is  certain  that  in  absence  of  fever,  sputum,  night  sweats,  chills 
and  loss  of  weight  an  active  process  is  not  probable  and  therefore  of  no  sig- 
nificance in  explaining  the  glycosuria.  Of  equally  little  importance  is  the 
nasal  obstruction  and  pharyngitis.  The  kidneys  are  anatomically  intact  as 


BLOOD   SUGAR  155 

far  as  the  physical  and  urinary  signs  are  concerned;  the  functional  tests  of 
these  organs,  however,  reveal  some  impairment  as  shown  by  a  slightly  dimin- 
ished phthalein  excretion  and  an  Ambard's  constant  barely  within  what  Has 
been  in  our  experience  the  upper  normal  figure.  The  connection  between 
such  a  diminished  kidney  function  and  a  possible  renal  diabetes  is  of  ex- 
treme interest.  The  small  eczematous  patch  in  this  case  could  not  be  re- 
garded as  a  complication  of  diabetes  mellitus,  since  the  hyperglycemia, 
which  is  the  direct  etiological  factor  of  such  a  condition,  was  lacking. 

The  urinary  nitrogen  was  determined  by  the  Kjeldahl  process,  the  am- 
monia according  to  Folin,  the  glucose  by  Benedict's  modification  of  Feh- 
ling's  method,  the  acid  bodies  by  Shaffer's  procedure.  The  method  of  Lewis 
and  Benedict  was  used  in  estimating  the  blood  sugar. 

Blood  sugar  determined  by  the  Lewis  and  Benedict  method  was  normal, 
although  urine  showed  glucose.  This  case  must  be  classed  as  one  of  true 
renal  diabetes.  There  was  slightly  diminished  phenolsulphonphthalein  ex- 
cretion, the  slight  elevation  of  Ambard's  constant  above  the  normal,  as 
well  as  the  glycosuria,  point  to  a  depressed  kidney  function.  The  absence 
of  any  further  subjective  or  objective  signs,  past  or  present,  leads  to  the 
conclusion  that  a  renal  glycosuria  is  an  interesting  anomaly,  but  of  no  im- 
portance to  the  organism  as  a  whole. 

The  question  of  prognosis  in  this  condition  is  the  most  important  problem 
which  remains  to  be  solved.  It  is  well  known  that  instances  of  true  dia- 
betes may  persist  for  years  without  changing  from  a  mild  to  a  severe  typo 
in  spite  of  the  lack  of  any  systematic  efforts  at  dietary  restriction,  thus 
resembling  renal  glycosuria.  It  is  not  certain  that  what  is  termed  renal  dia- 
betes may  not  develop  into  diabetes  mellitus,  especially  since  compara- 
tively little  is  known  of  the  early  stages  of  true  diabetes.  The  number  of 
cases  of  renal  glycosuria  thus  far  observed  has  been  small  and  none  of  them 
has  been  followed  for  a  sufficient  length  of  time  to  ascertain  whether  renal 
diabetes  is  congenital,  and  not  an  acquired  anomaly,  and  whether  it  may 
persist  indefinitely  without  changing  its  characteristics. 

The  intensity  of  renal  glycosuria  should  vary  with  the  degree  of  kidney 
permeability  to  dextrose.  With  a  threshold  only  slightly  depressed,  an 
intermittent  glycosuria  often  of  an  apparently  unexplained  origin  may  be 
present;  with  a  very  marked  depression,  changes  approximating  the  condi- 
tions found  in  phlorizin  poisoning  should  develop.  Intermediary  de- 
grees of  kidney  involvement  should  have  glycosuria  of  corresponding  in- 
tensity. If  the  present  ideas  of  the  relations  of  a  diminished  kidney  thresh- 
old for  sugar  are  true,  all  the  grades  of  intensity  indicated  should  be  dem- 
onstrated in  the  course  of  time. 

This  expresses  a  very  conservative  estimate  of  the  facts  at 
hand,  that  we  are  not  as  yet  justified  in  classifying  these  cases 
as  incipient  cases  of  diabetes  mellitus.  We  have  evidence  fur- 
nished by  blood  chemical  analyses  that  there  is  no  hyperglycemia 
in  these  cases.  Until  data  are  at  hand  showing  conclusively  that 
these  cases  without  increased  blood  sugar  but  with  glycosuria  do 
inevitably  lapse  into  hyperglycemia  with  the  concomitant  symp- 
toms of  diabetes  mellitus,  we  should  not  group  them  in  any  way 


156  BLOOD   AND   URINE    CHEMISTRY 

with  that  disease.  Blood  chemical  methods  indeed  alone  will 
furnish  the  evidence  which  will  eventually  place  these  cases  in 
their  proper  position. 

Before  passing  further  into  the  question  of  true  diabetes  mel- 
litus,  we  might  say  a  word  regarding  the  so-called  alimentary 
glycosuria.  One  formerly  distinguished  between  a  form  due  to 
the  ingestion  of  starch  and  that  due  to  the  ingestion  of  sugar 
(alimentary  glycosuria  e  saccharo}.  Naunyn34  attempted  to  dis- 
tinguish an  alimentary  glycosuria,  i.  e.,-one  due  entirely  to  the  in- 
gestion of  carbohydrates,  from  a  case  of  diabetes  mellitus,  by  a 
renal  test  meal.  Referring  to  this  question,  the  Journal  of  the 
American  Medical  Association35  states  in  part: 

"In  certain  individuals  the  capacity  of  utilizing  glucose  is  sup- 
posed to  be  lowered.  It  may  become  sufficiently  deficient  in  some 
instances  to  lead  to  so-called  alimentary  glycosuria  following  an 
overindulgence  in  carbohydrate  food.  In  a  healthy  person  it  is 
scarcely  possible  to  produce  glycosuria  by  the  lavish  administra- 
tion of  starchy  food,  since  the  liver  can  apparently  store  up  the 
excess  of  sugar  as  fast  as  it  is  produced  by  the  digestion  of 
starch  in  the  alimentary  canal  and  absorbed  into  the  portal  cir- 
culation. There  is  a  widespread  belief  that  when  preformed  glu- 
cose is  fed,  however,  the  assimilation  limit  may  be  more  readily 
reached  through  rapid  and  unduly  large  absorption  of  soluble 
carbohydrate.  It  may  become  very  important  to  ascertain  an 
incipient  functional  defect  of  this  sort,  since  it  may  be  the  indi- 
cation of  some  impending  diabetic  defect.  Accordingly  it  has 
been  customary  in  some  clinical  laboratories  to  ascertain  the  ' '  as- 
similation limit"  for  glucose  by  feeding  a  measured  quantity  of 
this  carbohydrate  or  some  other  sugar,  such  as  lactose  (milk 
sugar)  or  levulose  (fruit  sugar),  at  one  time,  and  watching  for 
a  transient  glycosuria  as  a  result.  To  the  examination  of  the 
urine  for  sugar  before  and  after  the  administration  of  the  car- 
bohydrate, the  analysis  of  the  sugar  content  of  the  blood  may 
now  easily  be  added. 

"Success  in  ascertaining  an  abnormal  tolerance  in  a  procedure 
of  the  sort  described  evidently  hinges  on  the  ability  to  postulate 
what  a  normal  functional  capacity  of  a  healthy  individual  in  such 

34i\aunyn:      Der  Diabetes  Mellitus,   Wien,   1906. 
"'Editorial :     Jour.  Am.   Med.  Assn.,   Sept.  2,   1916,  p.   748. 


BLOOD   SUGAR  157 

circumstances  should  be.  Lately  it  has  been  asserted  that  whereas 
the  'assimilation  limit'  is  low  in  diabetes,  it  is  abnormally  high 
in  certain  conditions  involving  a  malfuncton  of  some  of  the  en- 
docrine glands  notably  the  pituitary.  Taylor  and  Hulton,36  of  the 
Department  of  Physiological  Chemistry  at  the  University  of  Penn- 
sylvania, recently  remarked  that  by  common  consent,  rather  than 
by  accurate  experimentation,  the  limit  of  assimilation  of  glucose 
on  alimentary  administration  has  been  set  at  from  200  to  250 
gins,  on  the  empty  stomach.  From  this  -figure  downward  the  stu- 
dent of  diabetes  applies  the  test ;  from  this  figure  upward  the  stu- 
dent of  the  diseases  of  the  ductless  glands  applies  the  test.  The 
Philadelphia  investigators  have  made  a  number  of  observations 
on  healthy  medical  students,  to  whom  glucose  was  administered 
in  strong  solution  and  in  whom  blood  sugar  content  was  ascer- 
tained immediately  before  and  three  hours  after  the  sugar  was 
given.  As  a  result  it  is  clear  that  nearly  all  the  subjects  tolerated 
the  ingestion  of  200  gms.  without  exhibition  of  glycosuria.  Of 
nine  subjects  who  ingested  300  gms.,  only  three  displayed  gly- 
cosuria. Of  the  six  who  ingested  400  gms.,  only  two  had  gly- 
cosuria. In  five  instances  500  gms.  were  given,  with  the  pro- 
duction of  glycosuria  in  but  one.  Taylor  and  Hulton  regard  500 
gms.  as  the  physical  limit  of  ingestion,  except  in  one  who  has 
trained  to  the  test ;  it  is  very  large  in  bulk,  inclines  to  nauseate, 
and  apparent!}'  the  excess  is  not  rapidly  absorbed,  so  that  the 
test  probably  means  no  more  than  does  the  administration  of  400 
gms.,  which  is  usually  tolerated.  Polyuria  occurred  rarely,  and 
there  was  no  relationship  between  the  polyuria  and  glycosuria. 
Intestinal  disturbances  were  not  observed.  It  appears,  by  way 
of  contrast,  that  healthy  persons  cannot  ingest  300  gms.  of 
levulose  without  intestinal  disturbances.  Whether  this  result 
is  inherent  in  such  amounts  of  levulose,  or  is  due  to  some  impurity 
in  the  supposedly  pure  preparation  used,  could  not  be  determined. 
The  further  general  conclusion  was  drawn  that  even  the  larger 
quantities  of  sugar  do  not  markedly  influence  the  sugar  content 
of  the  blood.  In  the  majority  of  healthy  adult  males,  according 
to  Taylor  and  Hulton,  there  is,  apparently,  no  limit  of  assimila- 


38Taylor  and  Hulton:     Jour.   Biol.   Chem.,   1916,  vol.   xxv,  p.   173. 


158  BLOOD   AND   URINE    CHEMISTRY 

tion  of  glucose;  a  glycosuria  does  not  regularly  follow  the  largest 
possible  ingestions  of  pure  glucose. 

"Woodyatt,  Sansuin,  and  Wilder37  have  very  properly  pointed 
out  that  the  common  clinical  practice  of  estimating  sugar  toler- 
ance' as  the  number  of  grams  of  glucose  which  can  be  given  by 
mouth  all  at  once  and  just  fail  to  cause  glycosuria  will  not 
justify  any  tenable  conclusion  respecting  the  power  to  utilize 
glucose.  They  say: 

"  'When  sugars  are  administered  by  the  stomach,  the  length 
of  time  during  which  they  are  actually  brought  to  the  cells  must 
depend  on  the  motor  power  of  the  stomach  and  of  the  bowel  and 
on  the  rates  at  which  the  sugars  can  be  absorbed ;  and  even  when 
they  are  given  subautaneously  or  by  any  other  route  which  in- 
volves absorption  as  a  prelude  to  their  entering  the  blood,  the 
rates  at  which  they  enter  the  blood  will  depend  on  the  rates  at 
which  they  are  absorbed.  By  any  of  these,  but  especially  by  the 
oral  method,  the  actual  rate  of  entry  of  sugar  into  the  blood  and 
tissues  at  large  must  vary  with  a  wide  range  of  physical,  physio- 
logic and  pathologic  conditions  over  which  we  have  no  control; 
nor  will  it  ever  be  possible  by  such  methods  to  force  sugar  to 
enter  the  blood  any  faster  than  it  can  be  absorbed.  The  rate  of 
sugar  absorption  is  a  self-limited  thing,  for  when  a  certain  con- 
centration of  sugar  is  once  present  in  the  blood,  no  quantity  given 
by  mouth  or  subcutaneously  or  intraperitoneally  can  raise  it 
higher. ' 

' '  The  fact  that  prolonged  hyperglycemia  did  not  arise  in  Taylor 
and  Hulton's  trials  on  normal  persons  is  in  itself  an  indication 
that  one  could  scarcely  expect  marked  glycosuria  to  manifest 
itself.  It  has  been  found  that  a  man  weighing  70  kgs.,  when 
resting  quietly  in  bed,  may  receive  and  utilize  63  gms.  of  glucose 
by  vein  per  hour  without  glycosuria.  The  normal  tolerance  limit 
for  glucose,  expressed  as  a  velocity,  is  established  at  close  to  0.85 
gm.  of  glucose  per  kilogram  of  body  weight  hourly,  which  agrees 
approximately  with  what  Blumenthal  has  established  by  repeated 
small  intravenous  injections  in  animals.  It  can  easily  be  com- 
puted from  such  statistics  that  if  a  man's  resting  requirements 
were  3,000  calories  per  day,  he  could  thus  receive  double  what 

87Woodyatt,  Sansum,  and  Wilder:     Jour.  Am.  Med.  Assn.,  1915,  vol.  Ixv,  p.  2067. 


BLOOD   SUGAR  159 

he  needed,  or  enough  to  cover  the  caloric  expenditure  of  the 
same  man  during  the  heavy  physical  exertion.  In  view  of  these 
facts  perhaps  the  supposed  increased  'tolerance'  for  glucose  in 
some  of  the  ductless  gland  disorders  relates  to  a  gastrointestinal 
rather  than  a  metabolic  function." 

The  study  of  diabetes  mellitus  is  attracting  great  attention  at 
the  present  time,  mainly  because  of  the  advent  of  the  Allen  starva- 
tion treatment.  This  is  based  on  the  results  of  exact  animal 
experimentation.  It  is  bearing  the  richest  fruit  in  the  form 
of  excellent  therapeutic  results.  Diabetes  mellitus  is  said  to  be 
rapidly  increasing  in  incidence,  yet  this  may  simply  mean  that 
more  cases  are  discovered  now  that  routine  urine  analyses  are 
being  made.  Joslin  states  that  the  frequency  of  diabetes  in  the 
United  States  is  one  per  cent  of  all  individuals  (they  either  have 
the  disease  or  will  develop  it)  ;  also  that  the  frequency  of  diabetes 
in  a  community  may  be  the  index  of  the  intelligence  of  its  phy- 
sicians. The  routine  examination  of  the  urine  of  every  patient 
should  be  made  the  order  of  the  day,  not  altogether  because  we 
want  to  discover  diabetes,  but  because  we  want  to  know  some- 
thing about  other  conditions.  We  urge  that  the  Benedict  test 
for  sugar  be  given  the  preference  over  all  other  sugar  tests  of 
urine.  It  is  made  from  a  solution  that  is  stable,  and  besides,  shows 
sugar  at  times  when  Fehling's  test  does  not.  This  has  occurred 
in  our  experience  a  number  of  times.  The  routine  examination 
of  urine  does  not  mean  the  examination  of  the  single  specimen  in 
the  morning  before  breakfast.  It  may  be  surprising  to  some  to 
learn  that  at  this  time  sugar  is  often  absent  from  the  urine  of  a 
diabetic. 

If  one  must  rely  on  urinary  tests  and  not  utilize  the  blood  chemi- 
cal methods,  it  must  be  remembered  that  there  are  individuals 
with  a  lowered  power  of  assimilating  carbohydrates  who  secrete 
glucose  only  for  short  periods  in  the  day,  some  time  after  meals, 
and  then  only  in  small  quantities.  Even  true  diabetics  in  the  mild 
stage  are  often,  even  apart  from  diet,  free  from  glycosuria  for 
some  part  of  the  twenty-four  hours,  especially  in  the  morning 
before  the  first  meal.  Kleen38  stated  this  well  known  fact  as  fol- 
lows: "The  first  and  most  important  rule  is,  therefore,  never  to 

Diabetes  Mellitus,  P.   Blakiston's  Son  &  Co.,   1900. 


160  BLOOD   AND   URINE    CHEMISTRY 

use  for  a  test  a  single  specimen  of  urine  passed  when  the  pa- 
tient's stomach  is  empty,  before  the  first  meal  of  the  day.  The 
best  means  of  deciding  from  a  single  examination  of  the  urine 
whether  a  person  is  normal  or  not  in  this  respect  is  furnished  by 
a  sample  passed  an  hour  after  the  end  of  the  dinner.  At  this 
time  the  excretion  is  at  its  maximum." 

The  routine  examination  of  blood  chemically  will  some  day  be 
required  in  making  clinical  diagnosis.  To  recommend  this  at  the 
present  time  seems  Utopian,  yet  the  results  of  such  a  study  would 
certainly  repay  one  who  follows  it  out.  The  methods  which  have 
been  described  promise  accuracy  and  ease  of  performance  to  those 
qualified  to  undertake  this  work.  It  is  true  that  the  advantage 
of  the  Allen  treatment  lies  in  the  fact  that  the  dietetic  regime  may 
be  carried  out  without  elaborate  tests  of  blood  and  urine,  yet  a 
far  better  control  of  the  treatment  is  within  our  grasp  if  we 
resort  to  blood  chemical  estimation. 

The  author's  data  on  the  following  two  cases,  blood  and  urine 
of  which  they  carefully  studied,  will  demonstrate  the  discrepancies 
between  the  findings  in  urine  and  blood  of  diabetics.  The  first 
case,  Mrs.  R.,  was  under  observation  twenty-four  days,  during 
which  time  she  was  given  the  Allen  treatment.  This  was  a  young 
woman  of  twenty-three,  with  a  history  of  one  brother  dying  of 
diabetes.  She  had  developed  diabetes  mellitus  one  year  before 
coming  under  our  observation.  During  this  time  she  had  been 
under  various  dietetic  regulations  but  had  not  been  able  to  ac- 
complish much  in  the  way  of  permanently  relieving  herself  of 
diabetic  symptoms  or  of  glycosuria.  She  displayed  some  loss  of 
weight  and  polyuria  and  polydipsia.  At  the  time  of  the  first  ex- 
amination she  showed  0.360%  blood  sugar  and  was  excreting  78 
gms.  of  sugar  in  the  twenty-four  hour  specimen  of  urine.  She 
had  a  carbon  dioxide  combining  power  of  68,  with  a  large  amount 
of  acetone  and  diacetic  acid  in  the  urine.  She  was  watched  one 
week  before  beginning  the  Allen  treatment,  on  general  diet.  Dur- 
ing this  time  she  was  given  1/10  grain  parathyroid  three  times 
daily  for  certain  experimental  purposes.  During  this  week's  ob- 
servation, she  showed  a  marked  increase  in  the  amount  of  sugar 
in  the  urine,  but  the  amount  of  blood  sugar  did  not  materially 
change.  Her  chart  follows : 


BLOOD   SUGAR 


161 


CASE   OF  MRS.   "R,"   AGE   23   YEARS 


Date 

Wt. 

Kilos 

Diet 
Calor- 
ies** 

BLOOD 
ANALYSIS 

URINE  ANALYSIS* 

Sugar 
Per 
Cent 

C02 
Combin- 
ing 
Power  of 
Plasma 

Vol. 
C.  C. 

Sp. 
Gr. 

Sugar 
Grams 

Ace- 
tone 

Dia- 
cetic 
Acid 

Indi- 
can 

***9/19 
9/20 
9/21 
9/22 
9/23 
9/24 
***9/25 
9/26 
9/27 
9/28 
9/29 
9/30 
10/1 
10/2 
10/3 
10/4 
10/5 
10/6 
10/7 
10/8 
10/9 
10/10 
10/11 
10/12 
flO/13 
ftlO/30 

53.2 
51.4 
53.0 
52.3 
53.2 

54!i 
54.1 
54.0 
53.4 
53.6 
53.0 
52.3 
53.2 
54.1 
55.0 
55.7 
54.4 
54.2 
54.2 
54.4 
54.2 
53.9 
54.3 
54.9 

R 
R 
R 
R 
R 
R 
R 
F.  F. 
A.  T. 
A.  T. 
54 
234 
354 
504 
631 
823 
1131 
1305 
1525 
2023 
1719 
1845 
1883 
1819 
1859 

0.360 

68 

2600 
3160 
2600 
3000 
3200 
3500 
3650 
2200 
650 
800 
950 
1200 
720 
700 
850 
800 
1800 
1400 
1300 
950 
1100 
1400 
1250 
1100 
1200 

1037 
1047 
1040 
1042 
1040 
1042 
1040 
1040 
1020 
1022 
1027 
1024 
1026 
1028 
1026 
1029 
1011 
1010 
1011 
1015 
1014 
1014 
1011 
1016 
1015 
1022 

78 
126.4 
104 
150 
160 
175 
240.9 
110 
Neg. 
Neg. 
Neg. 
Neg. 

Seg- 

Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 

++++ 
++++ 
++ 
++ 
++ 
++ 
++ 
Trace 
++ 
++ 
++++ 
++++ 
++++ 
++++ 
++ 
++ 
V.F.T. 
V.F.T. 
Trace 
V.F.T. 

++++ 
++++ 
++++ 
++ 
++ 
++ 
+ 
Trace 
++ 
++ 
++++ 
++ 
++++ 
++++ 
++ 
++ 
V.F.T. 
V.F.T. 
Trace 
V.F.T. 

++ 
+ 
+ 
Xeg. 
+ 
+ 
+ 
Trace 
Neg. 
Neg. 
Trace 
+ 
++ 
++ 
Trace 
++ 
Trace 
Trace 
Neg. 
++ 
Trace 
++ 
++ 
++ 
+ 
Neg. 

6.36 

62 

0.120 

6.i20 

52 

6.129 

V.F.T. 
V.F.T. 

Neg. 

Neg. 
Neg. 
Neg. 

Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 

0.141 

*  +  +  +  +=  Large  amount. 

+  +=  Moderate  amount. 

+= Small  amount. 

V.F.T. = Very  faint  trace. 
**R= Regular  mixed  diet. 

F.F.  =  Fat-free  diet. 

A.  T.= Starvation. 


***During  above  period  patient  was  given 
1-10  grain  of  parathyroid  three  times  a 
day.  Note  increase  in  urine  sugar. 

t  Patient  left  hospital. 

ffUrine  received  by  mail. 


This  patient  has  been  heard  from  several  times.  She  is  now  tak- 
ing over  2,000  calories  and  sugar  has  reappeared  but  once  in  her 
urine.  Under  one  day's  starvation,  this  quickly  disappeared. 
Since  then  she  has  been  sugar-free.  No  opportunity  has  been  had 
since  to  obtain  her  blood  for  examination.  This  might  be 
termed  a  very  successful  issue  under  the  Allen  treatment. 

The  next  case,  that  of  Mr.  W,  represents  what  might  be  termed 
an  unsuccessful  case.  This  man,  aged  55  years,  married,  dis- 
played nothing  in  his  family  history  to  point  to  diabetes,  no 


162  BLOOD   AND   URINE    CHEMISTRY 

obesity,  gout  or  tuberculosis  in  father,  mother,  brothers,  sisters 
or  other  relatives.  He  was  an  occasional  drinker,  moderate  at 
venery,  formerly  did  a  good  deal  of  manual  labor,  sleeps  well. 
Three  and  a  half  years  ago  began  to  lose  weight  and  developed 
polyuria,  gradually  developing  polyphagia  and  polydipsia.  Sugar 
was  first  discovered  in  his  urine  three  years  ago  on  account  of 
having  consulted  his  physician  because  of  his  polyuria  and  loss 
in  weight.  So  far  as  etiological  factors  are  concerned,  he  had 
been  addicted  to  dietary  excesses.  He  gave  a  negative  Wasser- 
mann  and  Hecht-Gradwohl  test  for  syphilis,  had  never  had  any 
trauma,  had  occasional  pains  in  the  region  of  the  pancreas  but 
no  palpable  tumor.  There  was  no  disturbance  in  the  thyroids, 
no  symptoms  of  gout  (blood  uric  acid  was  normal  in  quantity), 
and  no  hypertension.  His  weight  on  coming  under  observation 
was  101  Ibs.,  height  5  feet  8  inches,  marked  loss  of  strength,  marked 
polyuria,  polyphagia,  pains  over  pancreatic  region,  had  numb- 
ness in  legs,  cramps  in  lower  legs,  had  lost  all  teeth  six  months 
before  (pyorrhea  alveolaris),  bowels  constipated,  had  occasional 
headaches,  coughed  frequently,  examination  of  lungs  disclosed 
evidences  of  beginning  tuberculosis  of  left  lung,  confirmed  micro- 
scopically. A  very  much  emaciated  man,  with  pale  visible 
mucosse,  thyroid  normal,  slight  delay  in  contraction  of  pupils, 
hearing  good,  breath  gave  acetone  odor,  arteries  soft.  Diagnosis: 
Diabetes  mellitus  and  pulmonary  tuberculosis.  His  urine  showed 
97.3  grams  sugar  in  twenty-four  hour  specimen  of  2950  c.c.  His 
blood  showed  0.280%  sugar.  (See  chart  on  page  163  for  full  facts 
of  this  study.) 

He  was  under  observation  forty-three  days.  He  was  tried  out 
on  the  Allen  treatment  but  responded  very  poorly.  The  highest 
amount  of  calories  he  could  take  without  producing  glycosuria 
was  1060 — clearly  insufficient  to  maintain  life.  He  was  in  a  state 
of  acidosis  at  the  very  beginning  of  his  observation,  showing  a 
carbon  dioxide  combining  power  of  but  50,  with  marked  amounts 
of  acetone  and  diacetic  acid  in  his  urine.  Every  attempt  was 
made  to  prevent  acidosis  and  to  keep  him  sugar-free  and  at  the 
same  time  give  him  sufficient  nourishment  to  support  life,  but  this 
was  never  successfully  consummated.  He  finally  left  the  hospital 
showing  a  persistent  hyperglycemia,  and  a  trace  of  sugar  under 
1060  calories  of  food.  He  was  apparently  doing  very  badly  un- 


BLOOD   SUGAR 


163 


CASE   OF   MR.   "W,"   AGE   55    YEARS 


Date 

Wt. 

Kilos 

Diet 
Calor- 
ies** 

BLOOD  ANALYSIS 

URINE  ANALYSIS* 

Sugar 
Per 
Cent 

CO2 
Combin- 
ing 
Power  of 
Plasma 

Vol. 
C.  C. 

Sp. 
Gr. 

Sugar 
Grams 

Ace- 
tone 

Dia- 
cetic 
Acid 

Indi- 
can 

10/1 
10/2 
10/3 
10/4 
10/5 
10/6 
10/7 
10/8 
10/9 
10/10 
10/11 
10/12 
10/13 
10/14 
10/15 
10/16 
10/17 
10/18 
flO/19 
10/20 
10/21 
10/22 
10/23 
10/24 
10/25 
10/26 
10/27 
10/28 
10/29 
10/30 
10/31 
11/1 
11/2 
11/3 
11/4 
11/5 
11/6 
11/7 
11/8 
11/9 

ttii/io 

ttll/11 
11/12 

46.6 
46.0 
46.0 
45.5 
45.0 
44.3 
44.6 
44.6 
42.5 
43.9 
42.4 
42.4 
42.2 
43.4 
43.4 
43.9 
43.4 
42.8 
43.4 
43.8 
43.4 
43.4 
43.2 
42.9 
43.6 
43.1 
42.8 
42.6 
42.8 
43.5 
42.9 
43.6 
43.0 
43.1 

isli 

42.6 
42.7 
43.0 
42.8 
43.4 
Patient 

R 
R 
F.  F. 
A.  T. 
A.  T. 
A.  T. 
A.  T. 
I.  S. 
A.  T. 
A.  T. 
A.  T. 
35 
220 
360 
462 
542 
734 
A.  T. 

3100 
2950 
1400 
800 
1400 
800 
950 
1200 
520 
740 
1600 
800 
900 
600 
1100 
950 
1600 
1100 
520 
1000 
650 
300 
750 
600 
850 
1000 
900 
850 
1100 
900 
950 
800 
1200 
870 
1050 
1200 
700 
550 
1200 
750 
1100 
1200 

1036 
1040 
1038 
1018 
1017 
1020 
1015 
1014 
1020 
1017 
1010 
1016 
1015 
1015 
1016 
1015 
1015 
1015 
1016 
1016 
1017 
1023 
1020 
1018 
1020 
1020 
1020 
1018 
1015 
1018 
1022 
1020 
1015 
1018 
1015 
1015 
1016 
1015 
1016 
1020 
1020 
1022 

+ 
+ 
++ 
++ 
+ 
+ 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Trace 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Trace 
V.F.T. 
Trace 
V.F.T. 
Neg. 
Neg. 
Neg. 
Trace 
Trace 

+ 
+ 
++ 
++ 
+ 
+ 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Trace 
Neg. 

Seg- 

Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Trace 
V.F.T. 
Trace 
V.F.T. 
Neg. 
Neg. 
Neg. 
Trace 
Trace 

Neg. 
+ 
Trace 
Trace 
Trace 
Trace 
Neg. 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 
Neg., 
Neg.  ' 
Trace 
Trace 
Neg., 
Trace 
+   ( 
Trace 
Trace 
Trace 
Trace 
+    I 
Trace 
Trace 
Trace 
Neg. 
Trace 
Trace 
+ 
Trace 
Trace 
Trace 
Trace 
Trace 
Trace 

0.280 

50 

97.3 
70.0 

+ 
+ 
Trace 
Trace 
Trace 
+ 
V.F.T. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Trace 
Trace 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Trace 
Neg. 
Trace 
Trace 
Trace 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Neg. 
Trace 
Neg. 
Trace 
Neg. 
Neg. 
Neg. 
V.F.T. 
Trace 
Trace 

0.200 

6.200 

49 

'6  '.266 

195 
140 
370 
478 
602 
A.  T. 
600 
629 
A.  T. 
A.  T. 
354 
472 
609 
866 
1038 
1044 
A.  T. 
751 
A.  T. 
A.  T. 
592 
939 
1058 
1060 
Left 

0.156 

55 

0.189 

54 

0.192 

Hospital. 

'  +  +  +  +=  Large  amount. 
+  += Moderate  amount. 
+= Small  Amount. 
V.F.T. = Very  faint  trace. 


**R=  Regular  mixed  diet. 

F.F.= Fat-free  diet. 

A.T.=  Starvation. 

I.S.= Intermittent  starvation. 
fPatient  fed  by  mistake. 
ttPatient  eating  outside. 


164  BLOOD   AND   URINE    CHEMISTRY 

der  the  treatment;  besides,  his  tuberculous  infection  seemed  to 
be  making  fast  inroads  upon  his  general  condition.  This  failure 
of  the  Allen  treatment,  of  course,  occurred  in  a  case  that  was 
both  an  advanced  diabetic  and  a  rapidly  advancing  pulmonary 
tuberculous  subject.  The  tuberculosis  infection  naturally  had 
impoverished  his  system  and  prevented  a  fair  trial  of  the  treat- 
ment. We  narrate  the  case,  however,  as  a  very  good  example 
of  a  study  of  blood  and  urine  in  complicated  diabetes  mellitus. 


CHAPTER  XXVI. 

ACIDOSIS. 

,  We  will  now  consider  acidosis,  its  cause,  its  symptomatology, 
its  significance,  its  recognition  by  blood  and  urinary  findings.  In 
acidosis  it  is  not  meant  that  the  reaction  of  the  blood  actually 
changes  from  its  alkaline  or  neutral  reaction  to  acid  reaction. 
This  is  impossible,  for  life  can  not  be  sustained  if  an  acid  condi- 
tion of  the  blood  occurs.  In  the  very  last  stages  of  life,  practi- 
cally in  extremis,  an  acid  condition  of  the  blood  occurs,  but  un- 
der no  other  circumstances. 

It  must  be  remembered  that  the  neutrality  of  the  blood  de- 
pends upon  the  mixture  of  carbonic  acid,  carbonates,  and  phos- 
phates in  the  blood  and  that  these  seem  to  remain  at  constant 
values  even  though  the  exogenous  source  of  alkalies  or  acids 
is  increased  or  diminished.  This  was  shown  by  Henderson.1  Car- 
bon dioxide  is  also  thrown  off  from  the  lungs  and  the  urine  in 
health  is  acid  in  reaction  ;  this  helps  in  maintaining  the  alkalinity 
of  the  blood.  The  physiology  of  the  respiratory  center  is  most 
interesting  for  when  the  amount  of  acid  increases  in  the  body, 
there  is  a  quick  stimulation  of  these  centers  with  the  result  that 
more  C02  is  thrown  out  and  the  acid  condition  of  the  blood  is 
prevented  from  assuming  larger  proportions.  Any  excess  of  acids 
induces  this  phenomenon.  When  the  acidity  of  the  blood  is 
threatening,  there  is  a  quick  call  on  the  ammonia.  It  is  only 
when  the  ammonia  is  being  used  up,  that  "acidosis"  supervenes. 
In  the  course  of  normal  metabolism  we  know  that  the  ammonia 
of  the  body  is  converted  into  urea  and  eliminated  as  such,  but  the 
supervening  acidosis  takes  up  some  of  this  ammonia  and  keeps  the 
blood  alkaline.  Application  of  principles  calling  for  an  estima- 
tion of  the  alveolar  carbon  dioxide  tension  of  course  gives  valu- 
able information  about  acidosis.  In  a  very  recent  publication, 
Marriott2  has  called  attention  to  a  simple  method  for  the  de- 


Henderson:     Ergeb.  d.  Physiol.,  1909,  vol.  viii,  p.  254;  Science,  New  York,   1913,  vol. 
xxxvii,  p.  389. 

Harriott:     Jour.  Am.  Med.  Assn.,  May  20,   1916.. 


166  BLOOD   AND   URINE    CHEMISTRY 

termination  of  this  tension.      We   shall  fully   cover   this  later. 

Howland  and  Marriott3  assert  that  the  term  is  loosely  used,  that 
acidosis  is  spoken  of  when  acetone  bodies  appear  in  the  urine. 
This  is  not  necessarily  true.  We  must  remember  that  the  regula- 
tors of  the  alkalinity  of  the  blood  are  (1)  sodium  bicarbonate,  oc- 
curring in  plasma  and  cells,  (2)  the  acid  and  alkaline  phosphates 
of  sodium  and  potassium  found  in  the  red  blood  ceUs,  and  (3) 
the  proteins.  Acid  in  the  shape  of  carbonic  acid  is  formed  in  the 
tissues.  Kespiration  lowers  the  concentration  of  C02  in  the 
lungs  and  allows  the  higher  concentrations  in  the  tissues  to 
escape  into  the  lungs  and  be  removed.  Concentration  is  highest 
in  the  tissues,  lower  in  the  blood,  and  lowest  in  the  lungs.  Hen- 
derson4 calls  carbonates  of  the  blood  the  first  line  of  defense 
against  acidosis.  Dyspnea  or  hyperpnea,  or  increased  pulmonary 
ventilation,  is  the  greatest  aid  for  the  liberation  of  carbon  dioxide 
from  the  body. 

A  second  line  of  defense  is  the  capacity  of  the  kidneys  to  ex- 
crete an  acid  urine  from  a  neutral  blood.  They  remove  acid 
phosphate  and  save  base  with  each  molecule  of  acid  phosphate 
that  they  excrete.  A  third  line  of  defense  is  furnished  by  the 
proteins.  Proteins  can  combine  with  appreciable  amounts  of 
either  acids  or  alkalies  without  undergoing  any  marked  changes 
in  reaction.  Another  line  of  defense  is  the  ammonia  of  the  body. 
The  body  can  neutralize  acid  by  producing  ammonia.  This  oc- 
curs at  the  expense  of  the  urea.  Aside  from  the  interest  we  have 
in  acidosis  as  part  and  parcel  of  our  study  of  diabetes  mellitus, 
acidosis  occurs  in  children  in  connection  with  other  conditions. 

Quoting  from  Howland  and  Marriott:5  "Even  when  no  evi- 
dence of  disease  can  be  detected  to  which  the  acidosis  can  be  re- 
ferred, acidosis  may  be  found.  For  instance,  a  boy  of  six  was 
suddenly  taken  ill  with  high  fever.  Inside  of  twelve  hours  he 
was  brought  to  the  hospital  with  great  dyspnea  of  the  air-hunger 
type.  Physical  examination  was  quite  negative  except  for  a 
purulent  otitis  media.  All  the  tests  made  indicated  acidosis. 
The  bicarbonate  of  the  blood  was  greatly  reduced.  The  reaction 

'Rowland,  John,  and  Marriott,  W.:  Bull.  Johns  Hopkins  Hosp.,  March,  1916,  vol. 
xxvii,  No.  301. 

4Henderson:     Am.  Jour.  Physiol.,   1908,  vol.  xxi,  p.   427. 

5Howland,  John,  and  Marriott,  W.:  Bull.  Johns  Hopkins  Hosp.,  March,  1916,  vol.  xxvii, 
No.  301. 


ACIDOSIS  167 

of  the  blood  had  shifted  markedly  toward  acidity  and  yet  the 
acetone  bodies  in  the  blood  were  not  greatly  increased.  The 
tolerance  for  alkalies  was  enormously  increased.  Though  he  took 
by  mouth  20  grams  of  soda  and  6  grams  by  rectum  without  vomit- 
ing or  diarrhea,  no  change  in  the  reaction  of  the  urine  was  pro- 
duced thereby.  But  the  alkalies  had  a  profound  influence  upon 
his  condition;  his  respirations  diminished  in  rapidity  and  depth, 
the  evidences  of  acidosis  to  be  obtained  by  the  various  tests 
rapidly  disappeared  and  he  made  an  uninterrupted  and  appar- 
ently complete  recovery;  for  he  now  seems  entirely  well  and  has 
been  so  for  six  months. 

"We  may  then  say  that  acidosis  is  not  an  uncommon  condition 
in  infancy  and  childhood;  that  while  it  is  especially  frequent  in 
the  severe  diarrheas  of  infancy,  it  may  appear  with  a  variety  of 
diseases,  and  sometimes,  apparently,  alone.  To  recognize  it  with 
older  children  is  not  very  difficult.  The  character  of  the  respira- 
tion is  usually  sufficient  to  arrest  one's  attention  and  one  or  two 
relatively  simple  laboratory  tests  will  quickly  determine  the 
question  one  way  or  the  other.  With  infants  who  are  irritable, 
restless  and  crying,  it  is  much  more  difficult  to  say  whether  hyper- 
pnea  is  present;  and  yet  with  them  it  is  most  important  to  make 
the  diagnosis  early,  for  the  reason  that  acidosis  is  such  a  fatal 
complication  of  diarrheal  disease  in  infancy.  Older  children  re- 
act promptly  and  often  permanently  to  alkali  therapy.  It  may 
be  possible  to  stop  the  clinical  and  laboratory  evidences  of  acidosis 
in  infants,  but  the  patients  usually  die.  Why  they  do  cannot 
be  determined  at  the  present  time.  Many  normal  processes  have 
undoubtedly  been  inhibited,  perhaps  permanently,  and  many  ab- 
normal ones  stimulated.  A  restoration  to  normal  conditions 
seems  nearly  impossible.  For  this  reason  we  should  not  wait 
until  acidosis  can  be  demonstrated.  From  the  beginning  we 
should  give  bicarbonate  of  soda  to  infants  with  severe  diarrhea 
in  sufficient  quantity  to  render  the  urine  alkaline  and  keep  it  so. 

"We  may  lay  it  down  as  a  general  maxim  that  as  hyperpnea 
indicates  acidosis,  so  hyperpnea  indicates  alkali  therapy,  and  this 
for  infants  or  older  children.  The  alkalies  may  be  given  by 
mouth,  by  rectum,  subcutaneously,  or  intravenously.  Vomiting 
and  diarrhea  frequently  render  their  administration  by  mouth  or 
by  rectum  out  of  the  question.  Then  one  of  the  other  methods 


168  BLOOD   AND   URINE    CHEMISTRY 

must  be  employed.  Intravenous  administration  is  the  method 
of  choice,  especially  when  rapidity  of  action  is  desired — and  with 
acidosis  rapidity  of  action  is  always  desired. 

"The  superior  longitudinal  sinus,  as  advised  by  Marfan,  Tobler 
and  Helmholz,  is  available  with  infants,  or  the  external  jugular 
or  femoral  veins.  With  older  children,  a  vein  in  the  arm  can  of- 
ten be  employed.  If  facilities  for  the  intravenous  injection  of 
alkali  are  not  at  hand,  the  injection  may  be  made  subcutaneously, 
with  care  that  the  bicarbonate  has  not  been  transformed  into  the 
carbonate,  else  severe  sloughing  of  the  tissues  may  result.  A  four 
per  cent  solution  is  usually  employed  for  intravenous  use  and  a 
two  per  cent  solution  for  subcutaneous  use.  The  quantity  to  be 
injected  depends  upon  the  size  of  the  child,  the  severity  of  the 
symptoms  and  the  effect  produced,  but  the  amount  is  always 
large.  It  must  be  given  until  the  urine  becomes  alkaline;  even  in 
infants  under  one  year,  as  much  as  10  gm.  in  24  hours  may  be 
required. 

"With  the  cases  of  acetone-body  acidosis  with  no  sugar  in  the 
urine  and  with  a  low  sugar  content  in  the  blood,  glucose  by  rec- 
tum, subcutaneously  or  intravenously,  seems  clearly  indicated  in 
addition  to  the  alkali.  With  all  forms  water  is  urgently  re- 
quired, especially  with  infants  who  are  dessicated  as  a  result  of 
the  vomiting  and  diarrhea. 

"Much  remains  to  be  learned  regarding  acidosis.  The  presence 
of  abnormal  acids  explains  the  origin  of  some  forms,  but  there  are 
others  that  are  in  nowise  understood.  Are  there  abnormal  acids 
whose  presence  has  not  been  detected?  Are  normal  acids  formed 
in  excess?  Are  bases  lost?  Does  the  kidney  fail  to  excrete  suf- 
ficient acid?  These  are  a  few  of  the  questions  at  present  unan- 
swered that  must  be  answered  before  our  knowledge  of  acidosis 
can  be  considered  in  any  way  complete.  Much  has  been  learned 
in  the  last  few  years ;  with  the  present  greatly  stimulated  interest 
in  the  subject,  we  may  confidently  expect  that  the  future  will 
provide  answers  to  many  of  the  questions  that  now  seem  obscure. ' ' 

Our  interest  in  acidosis  is  intimately  connected  with  the  dia- 
betic where  the  sugar  can  be  utilized  and  the  acetone  bodies  ac- 
cumulate in  the  blood.  The  study  of  the  hydrogen-ion  concen- 
tration of  blood  will  throw  light  on  diabetic  acidosis:  Marriott 
has  pointed  out  a  method  for  this  study  (see  page  66).  The  car- 


ACIDOSIS  169 

bon  dioxide  tension  of  alveolar  air  should  also  be  studied;  Mar- 
riott's method  determines  this  and  thus  estimates  the  degree  of 
severity  of  the  acidosis  and  the  results  of  the  treatment  of  the  same. 
This  is  a  very  excellent  way  of  arriving  at  such  a  conclusion,  but, 
it  must  be  remembered  as  Marriott  states  in  his  monograph,6  that, 
"Changes  in  the  pulmonary  epithelium  such  as  would  prevent  the 
air  in  the  lungs  from  coming  in  equilibrium  with  the  blood  in  the 
capillaries,  would,  of  necessity,  affect  the  composition  of  the 
alveolar  air.  Since  very  little  is  known  as  yet  regarding  the  exact 
effect  of  such  changes,  one  is  hardly  justified  in  drawing  conclusions 
regarding  acidosis  from  the  composition  of  the  alveolar  air  in 
patients  with  pulmonary  affections." 

The  neutralization  of  the  acidity  that  threatens  in  acidosis  oc- 
curs also  through  the  ammonia  reserve,  as  alluded  to  above. 
It  has  been  repeatedly  stated  by  writers  on  the  prevention  of  acid- 
osis that  the  consumption  of  fats  must  be  stopped,  in  fact,  in 
the  preliminary  preparation  of  a  patient  for  the  Allen  treatment, 
fats  must  be  excluded  so  as  to  prevent  or  lessen  the  chance  of 
acidosis  from  long-continued  fasting.  Why  is  this  true?  The 
metabolism  of  fats  will  easily  explain  this :  in  the  absence  of  the 
proper  carbohydrate  balance  or  tolerance  (which  is  the  situation 
that  exists  in  severe  diabetes)  the  substances  that  result  from  the 
cleavage  of  the  higher  fatty  acids  (such  as  stearin,  palmitin)  of 
fat,  are  transformed  into  oxybutyric  acid  and  diacetic  acid,  in- 
stead of  pursuing  the  normal  path  of  transformation  into  butyric 
acid.  There  is  no  further  oxidation.  Also  these  acids,  oxybutyric 
and  diacetic,  may  arise  from  certain  of  the  amino-acids,  leucine, 
tyrosine,  phenylalin,  which  occur  when  protein  is  split  up.  These 
organic  acid  derivatives  of  the  fat  and  protein  matter  of  the  body 
furnish  the  basis  for  the  formation  of  the  so-called  acetone  bodies 
which  are  acetone,  beta-oxybutyric  acid  and  diacetic  acid.  Their 
formulas  are  as  follows: 

CH3  -  CO  -  CH3  =  Acetone. 

CH3  -  CHOH  -  CH2  -  COOH  =  Beta-oxybutyric   acid. 

CH3 -  CO  -  CH2  -  COOH  =  Diacetic  acid  (aceto-acetic  acid). 

"When  these  bodies  appear  in  the  blood  in  excess  we  have  acidosis, 
but  it  must  again  be  stated  that  they  do  not  produce  an  acid  reac- 


"Marriott:     Jour.  Am.  Med.  Assn.,  1916,  vol.  Ixvi,  p.  1594. 


170  BLOOD   AND   URINE    CHEMISTRY 

tion  of  the  blood.  When  they  are  excreted  in  the  urine  we  speak 
of  ketonuria  or  acetonuria.  As  a  matter  of  fact  no  acetone  is 
eliminated  as  such  by  the  kidneys :  they  do  eliminate  diacetic  acid, 
but  from  this  acetone  is  formed  in  the  urine.  This  chemical  forma- 
tion is  easy  to  follow ;  it  simply  consists  in  the  diacetic  acid  throw- 
ing off  the  molecule  COOH,  resulting  in  acetone.  Emphasis  must 
be  laid  upon  the  fact  that  acidosis  does  not  occur  when  the  body 
is  easily  and  normally  burning  up  its  sugar.  It  is  when  it  can 
no  longer  do  so,  that  the  chemical  processes  already  explained  oc- 
cur. The  fats  under  normal  condition  are  burned  up  in  the  elab- 
oration of  the  carbohydrate  metabolism,  but  when  the  carbohy- 
drate metabolic  processes  are  in  abeyance,  then  the  fats  go  through 
their  imperfect  evolution  to  diacetic  acid  and  beta-oxybutyric 
acid  and  acetone,  i.  e.,  acidosis  then  occurs. 

We  have  called  attention  to  the  fact  that  the  ammonia  is  called 
upon  to  "suppress"  the  acidosis.  One  of  the  methods  for  de- 
termining the  ammonia  output  which  in  turn  will  guide  us  in 
estimating  the  degree  of  acetonuria,  is  to  determine  the  amount 
of  bicarbonate  of  sodium  necessary  to  render  the  urine  alkaline 
or  amphoteric.  Normally  from  5  to  10  grams  of  bicarbonate  of 
sodium  will  render  the  urine  alkaline.  In  mild  acidosis,  20  grams 
are  required;  in  severer  cases  from  30  to  40  grams;  and  in  ex- 
treme cases  40  grams  or  more.  In  coma,  when  urine  is  excreted, 
it  is  usually  impossible  to  neutralize  the  urine  or  make  it  ampho- 
teric, no  matter  how  much  sodium  bicarbonate  is  used.7 

Another  method,  however,  which  is  a  much  more  delicate  test 
for  acidosis  than  any  of  the  urine  tests  or  the  sodium  bicarbonate 
test  just  described,  is  the  estimation  of  the  carbon  dioxide  com- 
bining power  of  blood  plasma,  as  described  by  Van  Slyke.  Here 
we  have  a  ready  method  for  exactly  and  quickly  determining  the 
ability  of  the  patient's  blood  plasma  to  take  up  carbon  dioxide. 
When  the  ability  of  the  patient's  plasma  is  impaired  in  taking 
up  carbon  dioxide,  then  we  have  an  acidosis.  Thus  blood  plasma, . 
normally,  has  the  capacity  to  combine  with  65  per  cent  or  more 
of  the  carbon  dioxide,  which  can  be  thrown  into  it  in  the  form 
of  alveolar  air.  When  this  percentage  falls  below  50,  we  must 
consider  the  individual  in  a  state  of  acidosis.  This  method  is 
equal  in  efficiency  to  the  methods  of  determination  of  the  blood 

'Barker:     Monographic  Medicine,  vol.  iv,  p.  820. 


ACIDOSIS  171 

hydrogen-ion  concentration  of  Marriott  or  the  method  of  determina- 
tion of  the  carbon  dioxide  tension  of  alveolar  air.  The  character- 
istic readings  on  the  Van  Slyke  apparatus  are  anywhere  below  50 
in  marked  acidosis.  Thus  the  carbon  dioxide  combining  power  in 
a  case  of  diabetes  has  been  seen  to  drop  from  50  to  30.  The  ad- 
ministration of  alkalies  has  a  profound  influence  upon  it.  This 
brings  us  to  a  short  consideration  of  the  use  of  physical  and 
chemical  forces  in  combating  this  condition.  Inasmuch  as  the 
acetone  bodies  result  from  the  imperfect  and  incomplete  break- 
ing up  of  the  fat  molecule,  it  is  rational  to  interdict  the  use  of 
fats.  Secondly,  the  condition  occurs  as  a  result  of  imperfect  car- 
bohydrate metabolism.  The  glucose  is  not  being  burnt  up.  We 
try  to  burn  up  the  carbohydrates.  It  is  said  that  alcohol  assists 
in  the  burning  up  of  glucose,  and  therefore  should  be  tried.  Since 
alkaline  substances  taken  into  the  body  will  help  to  render  the 
urine  amphoteric,  we  must  quickly  throw  into  such  a  case  as 
much  sodium  bicarbonate  as  possible.  As  much  as  a  teaspoonful 
every  half  hour  in  water  should  be  given  to  a  patient  with  im- 
pending diaceturia  until  his  urine  becomes  amphoteric. 

Marriott,  Levy,  and  Kowntree8  have  described  their  method  for 
determination  of  the  hydrogen-ion  concentration  of  the  blood,  as 
given  on  page  66. 

It  might  be  advantageous  to  amplify  their  work  here  regarding 
the  variations  in  the  hydrogen-ion  concentration  of  the  blood. 
They  maintain  that  human  blood  as  it  exists  in  the  body  is  faintly 
alkaline  in  reaction,  that  is,  it  has  a  hydrogen-ion  concentration 
only  slightly  less  than  that  of  pure  water,  and  this  degree  of 
alkalinity  tends  to  be  maintained  even  when  considerable  quantities 
of  acids  are  produced  within  the  body,  or  are  introduced  from 
without.  Acidosis  may  be  recognized  in  various  ways,  by  an 
increase  in  the  ammonia  coefficient  in  the  urine,  decrease  of  carbon 
dioxide  tension  of  alveolar  air,  the  finding  of  abnormal  acids  in 
the  blood  and  urine,  increased  alkali  tolerance  and  by  dimin- 
ished titratable  alkalinity  of  the  blood  serum,  by  changes  in  the 
hemoglobin  dissociation  curve  and  by  actual  determination  of 
the  hydrogen-ion  concentration  of  the  blood.  A  change  in  the 
hydrogen-ion  concentration  of  the  blood  indicates  a  failure  of  the 


"Marriott,  Levy,  and  Rowntree:    Arch.  Int.  Med.,   1915,  vol.  xvi,  p.   388. 


172  BLOOD   AND   URINE    CHEMISTRY 

protective  mechanism  and  the  onset  of  acidosis.  It  is  in  this  con- 
nection that  the  determination  of  the  hydrogen-ion  concentration 
of  the  blood  according  to  the  technic  given  on  page  66  is  of 
value.  With  the  use  of  this  method,  a  series  of  bloods  from  normal 
and  pathologic  cases  were  studied  with  the  following  results: 

1.  Normal  individuals:  twenty-five  cases.  A.  Serum;  twenty- 
four  of  the  twenty-five  cases  read  between  7.6  and  7.8,  in  one  in- 
stance 7.9  was  the  record: 

pH  Cases 

7.6  4 
7.65  1 

7.7  5 
7.75  5 

7.8  9 

7.9  I 

B.  Whole  blood  (oxalated  by  collection  in  tubes  containing  a 
little  dry  powdered  sodium  oxalate,  free  from  carbonate)  ;  nine- 
teen determinations.     These  all  read  between  7.4  and  7.6 : 

pH  Cases 

7.4  3 
7.45  2 

7.5  4 
7.55  5 

7.6  5 

The  slightly  greater  acidity  of  whole  blood  as  compared  with 
serum  has  been  recognized  by  others  and  is  due  possibly  to  the 
fact  that  hemoglobin  and  especially  oxyhemoglobin,  behaves  as  a 
weak  acid. 

C.  Defibrinated  blood :    These  writers  used  early  in  their  work 
defibrinated  blood  run  in  parallel  series  with  serum  and  oxalated 
whole  blood.    No  additional  information  was  gained  by  using  de- 
fibrinated blood,   it  complicated  the  work  and   so   its   use  was- 
abandoned. 

2.  Miscellaneous  medical  cases  were  studied,  sixty-three  deter- 
minations in  52  cases,  comprising  the  following  diseases :  nephritis, 
"acute  and  chronic,    diabetes    mellitus,    myocardial    insufficiency, 
syphilis,  arthritis,  tuberculosis,  etc.     With  respect  to  the  serum 
of  these  cases,  sixty  of  the  sixty-three  determinations  read  be- 
tween 7.6  and  7.8.    With  whole  blood,  thirty-three  determinations 
gave  thirty-one  between  7.4  and  7.6. 

3.  Acidosis  cases  were  studied,  eight  cases  with  fifteen  determina- 


ACIDOSIS 


173 


tions.    The  general  conclusions  respecting  the  value  of  this  method 
of  estimating  acidosis  are  as  follows : 

A.  The  indicator  method  of  determining  hydrogen-ion  concen- 
tration is  made  applicable  to  blood  and  serum  by  utilization  of 
dialysis  through  a  collodion  membrane,  which  excludes  the  dis- 
turbing influences  of  color  and  of  proteins.  The  method  is  simple, 
accurate,  rapid,  and  well  adapted  for  clinical  work. 


Fig.    62. — Fridericia    apparatus    for    determination    of   carbon    dioxide    in   alveolar    air. 

B.  The  technic  consists  of  dialyzing  3  c.c.  of  blood  or  serum 
at  room  temperature  against  3  c.c.  of  0.8  per  cent  salt  solution  for 
five  minutes,  adding  an   indicator   and   comparing  with  colored 
standard  phosphate  mixtures  of  known  hydrogen-ion  concentra- 
tion. 

C.  Phenolsulphonphthalein  is  employed  as  the  indicator  in  this 


174  BLOOD   AND   URINE    CHEMISTRY 

method.  It  is  found  to  exhibit  easily  distinguishable  variations 
in  quality  of  color,  with  minute  differences  in  hydrogen-ion  con- 
centration between  the  limits  of  pH6.4  and  pH8.4. 

D.  Oxalated-  blood  from  normal  individuals  gives  a  dialysate 
with  a  pH  varying  between  7.4  and  7.6,  while  that  of  serum  ranges 
from  7.6  to  7.8. 

E.  Variations  from  these  figures  toward  the  acid  side  were  en- 
countered only  in  conditions  which  clinically,  and  from  the  stand- 
point of  laboratory  findings,  evidenced  an  acidosis. 

F.  In  a  small  series  of  clinical  acidoses,  the  serums  varied  from 
7.55  to  7.2  and  oxalated  blood  from  7.3  to  7.1.    In  experimental 
acidosis  in  dogs,  a  pH  of  6.9  was  encountered  in  both  serum  and 
blood  just  before  death. 

A  method  for  determination  of  carbon  dioxide  in  alveolar  air 
is  that  of  Fridericia  (Fig.  62).  This  method  does  not  involve  the 
use  of  expensive  apparatus,  can  be  transported  to  the  bedside, 
and  only  occupies  about  fifteen  minutes.  It  requires  the  coopera- 
tion of  the  patient  and  consequently  cannot  be  used  when  the  pa- 
tient is  in  coma,  but  when  this  occurs  the  Van  Slyke  and  urinary 
findings  will  suffice.  Fridericia9  described  his  method  in  1914. 
Horner10  describes  the  method  as  follows: 

"This  method  possesses  the  advantage  of  being  simple  and  in- 
volving the  use  of  apparatus  which  may  be  easily  transported  to 
the  bedside.  One  hundred  cubic  centimeters  of  alveolar  air  are 
collected  in  a  closed  chamber  and  then  cooled  from  the  tempera- 
ture of  the  body  to  that  of  the  room.  The  carbon  dioxide  in  this 
air  is  then  absorbed  with  a  20  per  cent  aqueous  solution  of  potas- 
sium hydrate,  thereby  creating  a  partial  vacuum,  which  in  turn 
is  equalized  with  water.  This  water  is  then  subjected  to  atmos- 
pheric pressure,  when  the  amount  of  carbon  dioxide  replaced  by 
water  can  be  read  in  percentage  of  atmospheric  air  by  reading 
the  height  in  centimeters  to  which  the  column  of  water  has  risen 
in  the  closed  100  c.c.  chamber.  This  percentage  may  be  changed 
to  millimeters  of  mercury  pressure  by  multiplying  the  difference 
between  barometric  pressure  at  the  time  of  the  test,  and 
this  varies  in  Boston  between  700  mm.  and  750  mm.,  and  the  ten- 
sion of  aqueous  vapor  at  37.5°  C.  which  is  48  mm.  mercury. 


9Fridericia:     Berl.  klin.  Wchnschr.,   1914,  p.  1268. 

10Horner:     Boston  Med.  and  Surg.  Jour.,  1916,  vol.  clxxv,  No.  5. 


ACIDOSIS  175 

This  will  make  a  factor  which  lies  between  718  and  702.  As  the 
reading  of  760  is  much  the  more  common  at  sea  level,  for  clinical 
purposes  the  factor  715  may  be  used  satisfactorily.  The  patient 
should  be  in  the  same  position  and  quiet  for  ten  minutes  prior  to 
the  performance  of  the  test. 

"After  a  normal  inspiration,  the  end  (A)  of  the  apparatus  is 
inserted  between  the  lips,  and  the  patient  is  instructed  to  expire 
forcibly  through  the  apparatus,  with  cocks  C  and  D  open,  so 
that  there  is  a  free  passage  from  A  to  B.  The  tube  remains  in 
the  mouth  throughout  the  entire  expiration  and  the  cock  C  is 
then  closed,  thus  retaining  between  cocks  C  and  D  the  last  100 
c.c.  of  expired  air.  (As  the  exchange  of  air  in  the  upper  respira- 
tory passage  is  200  c.c.  and  the  exchange  of  air  from  the  alveoli 
is  800  c.c.,  it  is  plain  that  with  any  care  at  all  a  sample  of  alveolar 
and  not  upper  respiratory  air  will  be  obtained.)  The  apparatus 
is  now  immersed  in  a  glass  tank  of  water  at  room  temperature 
and  allowed  to  remain  there  five  minutes.  The  best  way  to  ob- 
tain water  at  room  temperature  is  simply  to  keep  the  glass  tank 
in  the  room  with  the  patient  for  several  hours  before  the  test, 
though  with  an  ordinary  thermometer  one  can  easily  adjust  the 
temperature  of  the  water  to  that  of  the  room.  At  the  end  of  five 
minutes,  about  10  c.c.  of  20  per  cent  aqueous  solution  of  potas- 
sium hydrate  is  poured  into  the  apparatus  through  the  orifice  B. 
A  little  of  this  potassium  hydrate  will  leak  through  the  hole  in 
cock  D  to  chamber  CD.  Now  cock  D  is  turned  to  the  left  so  that 
chamber  CD  is  closed  and  chamber  BD  is  also  closed.  The  small 
amount  of  potassium  hydrate  in  chamber  CD  is  shaken  in  the 
chamber  for  a  moment.  Then  with  apparatus  in  upright  posi- 
tion, cock  D  is  turned  so  that  there  is  a  continuous  passage  from 
C  and  B,  and  the  amount  of  potassium  hydrate  which  will  run 
into  the  chamber  CD  is  allowed  to  do  so.  Now  cock  D  is  turned 
to  the  left  until  BDE  is  a  continuous  passage,  and  in  this  way 
potassium  hydrate  is  allowed  to  escape  into  the  water  tank. 
Chamber  CD  still  contains  2  or  3  c.c.  of  potassium  hydrate  solu- 
tion and  should  be  thoroughly  washed  with  this  solution.  Every 
point  in  the  surface  of  chamber  CD  must  be  touched  by  the  alka- 
line solution.  This  is  accomplished  by  shaking  very  thoroughly 
the  potassium  hydrate  in  chamber  CD.  The  apparatus  is  again 


176  BLOOD   AND   URINE    CHEMISTRY 

immersed  in  the  tank  of  water,  cock  D  is  turned  to  the  left  until 
water  rises  into  CD  through  EDC,  and  the  apparatus  left  in  the 
water  five  minutes.  At  the  end  of  this  time,  the  apparatus  is 
raised  until  the  bottom  of  the  meniscus  of  the  water  in  chamber 
CD  is  level  with  the  top  of  the  water  in  the  tank.  Now  cock  D 
is  turned  to  the  right  until  water  runs  through  EDB  to  the  level 
of  water  in  chamber  CD,  which  is  now  closed.  Then  cock  D  is 
turned  further  to  the  right  until  CDB  is  a  continuous  chamber. 
The  apparatus,  is  then  again  immersed  to  the  bottom  of  the  glass 
tank  and  the  water  in  the  arm  BD  of  the  apparatus  should  be  at 
the  same  level  with  the  water  in  the  chamber  CD  and  continuous 
with  it.  If  this  is  not  so,  then  the  amount  of  the  water  in  BD 
should  be  changed  until  it  reaches  the  height  of  the  column  of 
water  in  CD.  The  reading  is  now  taken  in  centimeters  of  the 
height  to  which  the  column  of  water  stands  in  CD,  and  this  is 
so  graduated  as  to  represent  the  percentage  of  C02  which  was 
absorbed  by  alkali  and  replaced  by  water.  This  completes  the 
test. 

"The  apparatus  is  prepared  for  the  next  test  by  opening  cock 
C  so  that  A  to  B  is  a  continuous  passage.  The  fluid  in  the  ap- 
paratus is  allowed  to  escape.  Orifice  B  is  put  under  the  faucet 
and  cold  water  allowed  to  run  through  the  apparatus,  taking  care 
to  shake  sufficiently  at  the  time  so  that  water  touches  all  of  the 
inside  of  the  apparatus.  Repeat.  Then  pour  through  orifice  B 
about  10  c.c.  of  4  per  cent  solution  boric  acid.  Rinse  the  ap-^ 
paratus  very  thoroughly  with  the  acid  so  that  there  shall  be  no 
alkali  remaining  adherent  to  its  sides.  Wash  again  with  cold 
water.  Leave  the  apparatus  so  that  orifices  A  and  B  are  down, 
thereby  allowing  any  water  in  the  apparatus  to  drain  out." 

From  the  above  it  will  be  seen  that  the  necessary  apparatus 
consists  of  the  Fridericia  appliance,  a  glass  tank  whose 
depth  is  equal  to  the  length  of  the  Fridericia  apparatus,  and  a 
wash  bottle  containing  4  per  cent  solution  of  boric  acid.  It  is 
convenient  *  to  add  an  indicator,  such  as  alizarin,  or  litmus,  to  the 
alkaline  and  acid  fluids. 

Of  the  several  methods  recommended,  the  Van  Slyke  method 
of  estimation  of  the  carbon  dioxide  combining  power  of  blood 
plasma  is  manifestly  preferable,  inasmuch  as  it  does  not  entail 
the  cooperation  of  the  patient  in  its  performance:  an  important 


ACIDOSIS  177 

point  when  dealing  with  unconscious  or  semiconscious  individuals. 
A  comparison  of  the  carbon  dioxide  tension  in  alveolar  air  by  the 
Plesh  method  with  the  amount  of  carbon  dioxide  in  the  venous 
blood  by  Van  Slyke's  method  has  recently  been  published  by 
Walker  and  Frothinghain.11  They  collected  the  air  for  the  method 
of  Plesh,12  as  modified  by  Higgins,13  in  the  apparatus  described 
in  detail  by  Boothby  and  Peabody.1*  In  this  method,  as  slightly 
modified  by  Boothby  and  Peabody,  the  patients  could  not  always 
cooperate,  yet  they  claim  consistent  results  followed.  In  their 
use  of  the  Van  Slyke  method  they  slightly  modified  the  technic, 
i.  e.,  instead  of  forcing  alveolar  air  into  the  separately  funnel 
from  the  operator's  lungs,  they  employed  a  separatory  funnel  of 
250  c.c.  capacity,  which  was  filled  from  a  spirometer  with  air  of  a 
known  carbon  dioxide  percentage.  Into  this  funnel  3  c.c.  of  the 
plasma  was  placed  and  shaken  for  two  minutes.  One  c.c.  of  this 
mixture  was  then  immediately  put  through  the  process  already 
described  on  page  59.  The  figure  obtained  after  being  corrected 
for  temperature  and  barometric  pressure  represented  the  number 
qf  milligrams  of  carbon  dioxide  in  1  c.c.  of  plasma.  Van  Slyke 
found  that  by  multiplying  this  figure  by  the  constant  35  he  ob- 
tained a  figure  comparable  to  that  obtained  for  the  carbon  dioxide 
tension  in  the  alveolar  air.  Their  observations  were  made  on  100 
different  cases  representing  thirty  different  types  of  disease.  A 
total  of  116  observations  in  all  were  made.  They  found,  for  in- 
stance, that  in  primary  anemia  the  carbon  dioxide  tension  in  the 
air  varied  in  different  cases  by  about  10  mm.  The  air  and  blood 
studied,  however,  did  not  vary  more  than  three  points.  In  a 
group  of  cases  of  Graves 's  disease,  the  carbon  dioxide  tension  was 
slightly  higher  than  the  blood  combining  power,  and  in  a  few 
the  difference  was  considerable.  In  typhoid  fever  the  results  were 
practically  identical.  In  two  cases  of  lung  abscess  the  results 
were  similar.  In  cases  of  chronic  nephritis  the  results  were  prac- 
tically alike.  It  was  found  that  when  the  carbon  dioxide  tension 
was  lowered  in  chronic  nephritis,  the  combining  power  of  the 
blood  plasma  was  similarly  lowered.  In  three  cases  of  syphilis 


"Walker   and    Frothinghain:      Arch.    Int.    Med.,    Sept.    15,    1916,    vol.   xviii,    No.    3,    pp. 
304-312. 

"Plesh;     Ztschr.  f.  exper.   Path.  u.   Therap.,  1909,  vol.  iii,  p.  380. 

Dn,   1915,  p.   168,  pub.  403. 
vol.  Kin,  p.  225. 


12Plesh;  Ztschr.  f.  exper.  Path.  u.  Therap.,  190$ 
13Higgins:  Carnegie  Inst.  of  Washington,  1915, 
"Boothby  and  Peabody:  Arch.  Int.  Med.,  1914, 


178  BLOOD   AND   URINE    CHEMISTRY 

the  results  were  identical.  Except  in  one  case  of  cardiac  dis- 
ease with  considerable  emphysema,  the  studies  were  alike  in  cases 
of  chronic  cardiac  disease.  Even  in  cases  of  pneumonia  where 
the  respirations  were  hurried  and  the  patients  could  not  co- 
operate very  well,  the  results  were  about  the  same.  In  acute  articu- 
lar rheumatism  there  were  similar  findings  except  that  there  was 
a  difference  in  one  case  of  as  much  as  thirteen  points.  In  five 
out  of  six  cases  of  diabetes  the  air  and  the  blood  showed  practi- 
cally the  same  carbon  dioxide  tension.  The  sixth  one  showed  a 
more  marked  variation,  yet  both  determinations  showed  evidence 
of  an  acidosis,  so  that  the  variation  in  this  case  would  not  have 
been  at  all  misleading.  It  is  interesting  to  note  that  in  all  the 
cases  of  diabetes  which  showed  acidosis,  the  blood  was  lower  in 
carbon  dioxide  than  the  air.  In  other  diseases  the  same  story 
was  told.  In  summing  up  the  116  observations,  the  carbon  dioxide 
tension  by  the  Plesh  method  corresponded  with  that  estimated  in 
the  blood  by  the  Van  Slyke  method.  But  little  choice  from  the 
standpoint  of  accuracy  can  be  offered  with  these  two  methods, 
but  we  recommend  the  Van  Slyke  method  as  being  the  simpler. 

Summarizing,  it  may  be  stated  that  fasting  for  a  normal  in- 
dividual is  apt  to  be  followed  by  acidosis  quicker  than  for  a  dia- 
betic subject.  This  is  admirably  seen  in  the  Allen  treatment, 
where  fasting  is  not  followed  by  acidosis,  whereas  in  a  normal  in- 
dividual in  a  few  days  he  would  begin  to  show  the  characteristic 
signs  of  blood  and  urine  of  acidosis  and  ketonuria.  The  body  has 
certain  safeguards  against  acidosis  which  are,  the  removal  of 
acids  from  the  blood  through  the  lungs,  the  pulmonary  action 
being  increased  by 'the  stimulation  of  excessive  acidity,  and  again 
the  fact  that  there  is  a  reaction  between  the  molecule  of  disodium 
phosphate  and  a  molecule  of  acid  by  which  the  sodium  carbonate 
of  the  blood  is  conserved  with  the  elimination  of  large  quantities 
of  acid.  The  amount  of  alkali  in  the  body  acts  as  a  factor  of  safety 
against  acidosis,  in  the  form  of  sodium  and  potassium  as  well  as 
the  calcium  and  the  magnesium  of  bones.  We  will  call  attention 
later  on  to  this  point  in  relation  to  the  mineral  metabolism  of  the 
urine.  The  factor  of  ammonia  in  the  body  must  again  be  em- 
phasized. This  is  due  to  the  fact  that  the  body  can  excrete  nitro- 
gen in  the  form  of  ammonia  from  the  proteins,  thereby  convert- 
ing some  of  the  endogenous  protein  whose  normal  destiny  is  urea 


ACIDOSIS  179 

into  ammonia.  It  must  be  remembered  that  one  gram  of  am- 
monia can  neutralize  five  times  as  much,  beta-oxybutyric  acid 
as  one  gram  of  sodium  bicarbonate.15  The  retention  of  the  al- 
kalinity of  the  blood  is  possibly  best  explained  in  Rowland's  own 
language.16  ''The  important  constituents  of  the  blood  so  far  as 
the  regulation  of  the  reaction  is  concerned  are  (a)  sodium  bi- 
carbonate, occurring  both  in  the  plasma  and  in  the  cells,  (b)  the 
acid  and  alkaline  phosphates  of  potassium,  found  almost  entirely 
within  the  red  blood  cells,  and  (c)  the  proteins. 

"  Considering  the  blood  first  as  a  solution  of  bicarbonates :  a 
large  amount  of  acid,  carbonic  acid,  is  constantly  being  formed 
in  the  tissues.  It  must  be  removed  by  the  lungs,  but  first  it  must 
be  transported  to  the  lungs  by  the  blood.  This  stream  of  acid 
which,  with  an  adult,  in  the  course  of  the  day,  is  the  chemical 
equivalent  of  several  hundred  cubic  centimeters  of  concentrated 
hydrochloric  acid,  is  sufficient  to  render  acid  any  ordinary  solu- 
tion and  keep  it  permanently  acid.  If  this  should  happen  in  the 
blood,  life  would  of  course  be  impossible,  but  owing  to  the  laws 
that  govern  the  reaction  of  solutions  of  weak  acids  and  their  salts, 
the  solutions  of  bicarbonate  are  able  to  take  up  a  quantity  of  the 
acid,  carbon  dioxide,  without  appreciably  undergoing  a  change 
in  reaction.  Thus  there  can  be  transported  from  the  tissues  to 
the  lungs  and  so  continuously  eliminated  from  the  body,  a  very 
large  amount  of  acid.  This  steady  escape  of  acid  is  accomplished 
with  no  harm  and  with  no  strain  upon  the  organism.  The  respira- 
tory center  is  adjusted  to  assist  in  the  removal  of  the  carbon 
dioxide.  If  there  were  no  respirations  and  circulation  were  con- 
tinued, eventually  the  carbon  dioxide  concentration  would  be  the 
same  in  the  tissues,  in  the  blood  and  in  the  air  and  in  the  pul- 
monary alveoli. 

"But  the  respirations  lower  the  concentration  in  the  lungs 
and  thus  allow  the  carbon  dioxide  to  escape  from  the  tissues  where 
the  concentration  is  highest  by  the  blood  where  the  concentration 
is  lower,  to  the  air  in  the  lungs  where  the  concentration  is  low- 
est. The  respiratory  center  is  extraordinarily  sensitive  to  the 
slightest  alteration  in  the  reaction  of  the  blood  toward  the  acid 
side,  so  that  an  increased  production  of  carbon  dioxide  in  the 


"  15Joslin:     I<oc.  cit.,  page  137. 
"Rowland:     Bull.  Johns  Hopkins  Hosp.,   1916,  vol.   xxvii,  p.  63. 


180  BLOOD   AND   URINE    CHEMISTRY 

tissues,  such  as  occurs,  for  instance,  with  muscular  exercise,  and 
the  resultant  slight  excess  in  the  blood  is  answered  by  an  increased 
ventilation  of  the  lungs  which  removes  the  carbon  dioxide,  thereby 
bringing  the  reaction  of  the  blood  back  to  normal.  Other  acids, 
whether  formed  in  the  body  or  introduced  from  outside,  produce  a 
similar  effect.  They  displace  the  carbonic  acid  from  the  sodium 
bicarbonate  and  set  the  carbon  dioxide  free.  This  excess  of  car- 
bon dioxide  is  removed  by  the  increased  pulmonary  ventilation 
leaving  a  neutral  salt,  sodium  oxybutyrate,  or  chloride,  or  what 
not,  to  be  removed  by  the  kidneys.  Such  a  mechanism  allows  rela- 
tively huge  amounts  of  abnormal  acids  to  be  at  once  rendered 
innocuous  and  removed;  for  instance,  NaHC03  +  HCL  =  NaCL  + 
H20  +  C02.  The  hydrochloric  acid  is  neutralized  and  the  result- 
ant sodium  chloride  is  removed  by  the  kidneys  while  the  carbon 
dioxide  is  given  off  by  the  lungs. 

"Henderson  calls  the  carbonates  of  the  blood  the  first  line  of 
defense.  Thus,  dyspnea,  more  properly  hyperpnea  or  increased 
pulmonary  ventilation,  under  abnormal  circumstances,  is  an  agent 
of  the  greatest  value  in  ridding  the  body  of  carbon  dioxide  and 
thus  keeping  the  reaction  within  normal  limits.  It  may  also  be 
remarked  that  hyperpnea  is  the  best  of  all  the  evidences  of  acidosis 
to  be  obtained  by  physical  examination  alone.  It  may  almost  be 
said  that  hyperpnea  means  acidosis. 

"If  the  bicarbonates  of  the  plasma  were  the  only  method  of 
defense  of  the  body,  the  organism  would  succumb  to  acidosis  as 
soon  as  the  bicarbonate  was  depleted  by  the  excretion  of  neutral 
salts  through  the  kidneys ;  every  molecule  of  an  acid  would  rob 
the  body  of  a  molecule  of  bicarbonate.  The  second  mechanism 
here  comes  into  play  and  is  that  by  which  acids  may  be  removed 
leaving  behind  part  of  the  base  with  which  they  have  been  com- 
bined, this  base  being  available  for  further  neutralization.  The 
elimination  is  by  way  of  the  kidneys.  These  have  the  capacity  to 
excrete  an  acid  urine  from  a  nearly  neutral  blood.  They  remove 
acid  phosphate  and  save  base  with  each  molecule  of  acid  phos- 
phate that  they  excrete.  Thus,  although  alkali  is  eliminated  in 
the  urine,  it  is  much  less  than  would  be  the  case  without  this 
specialized  kidney  activity,  and  can  readily  be  replaced  under 
normal  circumstances  by  the  alkali  of  the  food.  For  instance, 
with  the  introduction  of  a  foreign  acid— Na2HP04  +  HCL  = 


ACIDOSIS  181 

NaCL  -fMaH2P04 — the  hydrochloric  acid  is  neutralized,  the  sodium 
chloride  and  acid  sodium  phosphate  are  excreted  by  the  kidneys 
or  the  following  reaction  may  take  place — Na2HP04  +  H20  +  C02 
=  NaH2P04  +  NaHCO3.  By  this  method  the  sodium  bicarbonate 
reserve  of  the  body  is  renewed. 

"Henderson  and  Palmer  showed  the  magnitude  of  alkali  spar- 
ing very  prettily  by  titrating  with  alkali  the  acid  urine  back  to 
the  normal  reaction  of  the  blood.  The  alkali  spared  was  found  in 
normal  subjects  to  vary  in  terms  of  tenth  normal  alkali,  between 
200  and  800  c.c.  This  is  equivalent  to  saying  that  the  kidneys 
eliminate  from  200  to  800  c.c.  of  tenth  normal  acid  in  24  hours.'' 

A  very  authoritative  discussion  on  the  question  of  fat  in  dia- 
betes, in  relation  to  acidosis  particularly,  is  that  of  F.  M.  Allen, 
in  his  lecture  before  the  Harvey  Society  of  New  York,17  entitled 
"The  Role  of  Fat  in  Diabetes."  He  showed  the  development 
of  the  methods  of  these  problems  by  means  of  the  new  blood 
chemical  tests  which  we  have  already  described  in  Part  I  of 
this  work.  He  said  truly  that  it  was  a  fine  tribute  to  American 
science  that  every  one  of  these  tests  was  devised  or  perfected  by 
an  American  investigator.  Finally,  the  possibility  of  better 
study  of  the  problems  of  diabetes  was  greatly  increased  by  the 
ability  to  reproduce  in  dogs  conditions  almost  identical  with 
those  encountered  in  human  diabetes.  This  could  be  done  by  the 
surgical  removal  of  a  large  proportion  of  the  pancreas,  leaving 
the  remainder  in  communication  with  the  intestine  through  the 
pancreatic  duct.  This  operation  rendered  the  dogs  diabetic  and 
yet  retained  their  digestive  functions  through  the  preservation  of 
the  pancreatic  secretion. 

The  first  point  in  the  problem  of  the  role  of  fat  in  diabetes 
was  that  of  lipemia.  This  condition  wras  almost  a  constant  find- 
ing in  severe  human  diabetes  and  might  be  present  to  a  slight 
degree  even  in  very  mild  cases.  The  same  was  found  to  be  true 
in  partially  depancreatized  dogs.  Further,  diabetes  in  man  £ind 
the  partial  depancreatization  of  dogs  were  the  only  conditions  in 
which  a  high  degree  of  lipemia  was  found.  The  fat  might  be 
present  in  the  plasma  of  severe  cases  in  either  man  or  dog  in 
amounts  up  to  15  per  cent  or  over,  and  the  ability  to  produce  the 


ITAllen,  F.  M.:     New  York  Med.  Jour.,  Nov.   18,  1916. 


182  BLOOD   AND   URINE    CHEMISTRY 

condition  in  the  latter  afforded  ideal  conditions  for  the  study  of 
its  causation  and  significance.  It  had  long  been  believed  that  li- 
pemia  was  due  to  a  diminution  in  the  lipase  present  in  the  blood, 
but  this  could  now  be  stated  to  be  incorrect  and  we  could  safely 
regard  the  lipase  as  quite  a  negligible  factor.  It  had  been  shown 
in  experimental  dogs  that  lipemia  varied  in  degree  largely  with  the 
digestive  power  of  the  animals,  that  the  fat  was  derived  in  great 
measure  from  the  food  fats,  and  that  lipemia  could  be  controlled 
largely  by  feeding.  The  fat  in  the  blood  was  chiefly  neutral  fat 
with  a  considerable  proportion  of  cholesterol,  which  ran  parallel 
to  the  former,  and  a  small  amount  of  lecithin. 

As  to  the  causation  of  lipemia,  experiments  on  the  partially  de- 
pancreatized  dogs  made  it  possible  to  say  definitely:  1.  Lipemia 
was  not  due  to  the  occurrence  of  hyperglycemia.  2.  It  was  not 
due  to  the  absence  of  carbohydrate  or  to  the  loss  of  sugar.  3.  It 
was  not  due  to  the  presence  of  acetone  bodies  or  to  the  change  in 
the  reaction  of  the  blood.  4.  It  could  not  be  produced  by  simple 
overfeeding  with  fat.  Its  exact  cause  is  as  yet  unknown,  but 
recent  studies  in  the  author's  laboratory  seem  to  point  to  its 
being  related  in  some  way  to  the  condition  of  the  cells  in  the 
pancreas,  and  evidence  is  accumulating  which  indicates  that  there 
may  be  an  internal  secretion  of  that  gland  which  is  directly  con- 
cerned with  the  production  of  lipemia. 

The  second  problem  in  the  role  of  fat  in  diabetes  is  con- 
cerned with  acidosis.  Before  entering  into  its  discussion,  it  is 
necessary  to  have  a  clear  understanding  of  what  was  meant  by 
acidosis.  In  the  author's  opinion  the  term  should  be  restricted 
to  the  original  definition  given  by  Naunyn,  which  stated  that  its  one 
constant  characteristic  was  the  occurrence  in  the  blood  of  an  ab- 
normal amount  of  beta-oxybutyric  acid  and  acetone  bodies.  Con- 
trary to  the  present  misuse  of  the  term  it  had  nothing  to  do 
with  a  simple  displacement  of  the  reaction  of  the  blood,  and  con- 
ditions with  diminished  alkalinity,  increased  carbon  dioxide  ten- 
sion, increased  hydrogen-ion  concentration,  and  reduction  of  the 
"buffer"  salts  should  not  be  classed  as  acidosis,  since  such  a 
classification  led  to  confusion. 

Here,  as  in  lipemia,  the  precise  ultimate  cause  of  acidosis  is  not 
known.  It  was  fairly  certain,  however,  that  fat  played  an  im- 


ACIDOSIS  183 

portant  role  in  its  production  and  that  the  acids  were  produced 
largely  in  the  muscles  and  liver — organs  in  which  fat  was  burned. 
It  was  not  yet  known  what  proportion  of  fat  could  be  burned 
without  the  production  of  acidosis  in  subjects  with  diabetes,  or 
what  proportion  of  carbohydrate  was  required  to  prevent  the  de- 
velopment of  acidosis.  It  could  be  stated  positively,  however,  that 
acidosis  was  not  necessarily  due  to  a  lack  of  carbohydrate.  If  it 
was  not  possible  to  state  the  ultimate  causes  of  acidosis  at  least 
the  study  of  the  partially  depancreatized  dogs  had  made  it  pos- 
sible to  gain  an  insight  into  some  of  the  more  remote  causes. 

It  was  found  that  acidosis  could  be  produced  in  such  dogs  in 
three  ways,  all  in  complete  imitation  of  the  conditions  encoun- 
tered in  man.  First,  it  could  be  produced  by  following  the  plan 
adopted  in  the  usual  clinical  treatment  of  human  diabetes,  namely, 
by  giving  a  diet  of  high  caloric  value  and  high  fat  content.  If 
an  experimental  dog  with  diabetes  be  made  to  hold  or  to  gain 
weight — which  is  the  practice  in  man — fat  must  be  introduced 
into  the  dietary  and  calories  must  be  crowded.  One  of  two  things 
soon  happens  in  the  dog;  either  he  begins  to  vomit  and  suffer 
from  diarrhea  with  loss  of  .weight  and  refusal  of  the  food,  or,  if 
the  feeding  is  forcibly  continued,  his  metabolism  breaks  down. 
When  the  latter  occurs  true  acidosis  develops  and  a  fatal  dia- 
betic coma  quite  similar  to  that  in  man  ensues.  Such  a  diabetic 
coma  can  be  produced  in  these  animals  while  they  are  thus  kept 
on  a  full  diet,  and  this  is  just  what  occurs  in  human  beings. 
Secondly,  if  the  treatment  employed  in  moderate  human  cases  be 
applied  to  these  dogs,  the  same  results  will  ensue  as  in  the  first 
case.  This  is  the  fattening  treatment  which  is  marked  by  a  re- 
duction in  the  intake  of  protein  and  the  administration  of  fat. 
These  dogs  look  extremely  well,  but  they  go  on  to  a  fatal  acidosis. 
The  third  way  is  that  in  which  the  animals  are  kept  free  from 
glycosuria  through  the  administration  of  a  diet  very  low  in  car- 
bohydrates and  consisting  mainly  of  fat  and  protein.  This  form 
of  diet  is  also  often  prescribed  for  man. 

In  both  man  and  in  these  animals  if  the  condition  has  not 
gone  too  far  the  acidosis  may  be  checked  by  the  introduction  of 
a  period  of  fasting,  but  if  the  diet  is  restored,  the  downward 
progress  will  continue.  In  severe  cases — human  or  animal — the 


184  BLOOD   AND   URINE    CHEMISTRY 

fasting  may  at  first  increase  the  acidosis,  but  if  the  fasting  is  re- 
peated with  periods  of  return  to  a  properly  adjusted  diet,  it  is 
usually  possible  to  produce  an  immunity  to  the  fasting  acidosis 
and  an  ultimate  recovery  of  very  marked  degree.  These  observa- 
tions, along  with  others,  the  details  of  which  cannot  be  given,  all 
point  to  the  existence  of  some  specific  internal  function  of  the 
pancreas  which  is  concerned  with  the  production  of  acidosis. 
They  also  show  that  an  alteration  in  the  reaction  of  the  blood  is 
not  the  cause  of  death  in  acidosis,  for  the  blood  may  be  kept  nor- 
mal in  reaction  by  the  proper  administration  of  alkalies,  and  yet 
the  man  or  the  animal  may  die  of  diabetic  coma  and  typical 
acidosis. 

If  periods  of  fasting  are  properly  introduced  and  the  diet  is 
adjusted,  it  is  possible  to  keep  the  human  or  animal  patient  in  a 
Condition  of  physical  comfort  and  fair  health  for  long  periods 
o'f  time,  and  ultimately  to  increase  his  tolerance  for  foods  to  a 
great  extent.  It  was  also  pointed  out  that  the  craving  for  car- 
bohydrate seen  in  many  diabetics  was  not  due  to  "original  sin,'* 
but  was  a  physiological  demand  for  that  food  element  which  does 
the  most  perhaps  to  control  the  development  of  acidosis.  The  same 
craving  was  to  be  observed  in  an  intense  degree  in  the  dt»gs  suffer- 
ing from  acidosis. 

The  last  point  to  be  discussed  by  Allen  was. the  value  of  fat  in 
the  dietary  of  diabetics,  and  it  was  shown  that  fat  unbalanced  by 
other  food  constituents  was  a  poison.  The  essence  of"  these  observa- 
tions was  to  show  that  it  was  necessary  to  preserve  a  natural  bal- 
ance between  fats  on  the  one  hand  and  protein  and  carbohydrate 
on  the  other  if  dangerous  complications  were  to  be  avoided — 
especially  acidosis  and  coma. 

The  net  results  of  the  observations  pointed  to  the  absolute  neces- 
sity for  clearing  up  the  lipemia  of  diabetes ;  to  the  need  of  a  proper 
appreciation  of  the  importance  of  fat,  unbalanced  by  other 
foods,  in  the  production  of  acidosis;  and  finally  to  the  most  im- 
portant fact  of  all,  namely,  that  in  diabetes  there  was  a  deficient 
assimilative  function  and  that,  efforts  to  maintain  the  body  weight 
by  high  calory  feeding  would  soon  lead  to  an  exhaustion  of 
whatever  function  remained  to  the  patient.  The  true  lesson  to 
be  learned  was  that  it  was  not  fat  alone,  not  protein  alone,  and 


ACIDOSIS  185 

not  carbohydrate  alone  which  was  the  source  of  danger,  but  that 
it  was  a  disturbed  balance  between  all  three  combined  with  an 
overtaxing  of  the  patient's  assimilative  powers  which  led  to  the 
downward  progress  of  diabetics  under  the  usual  plans  of  dietetic 
regulation.  Depending  upon  the  severity  of  the  case,  the  load  on 
his  assimilative  function  should  be  lightened ;  if  he  had  acidosis 
he  should  be  starved,  once  or  repeatedly,  until  his  assimilative 
function  could  be  restored ;  and  his  diet  should  be  kept  within 
his  assimilative  capacity.  If  such  a  plan  were  followed,  the  ma- 
jority of  patients  would  live  in  comfort,  and  a  large  proportion 
would  ultimately  show  a  decided  increase  in  the  extent  of  their 
assimilative  capacities. 

In  connection  with  the  blood  chemical  methods  for  estimating 
acidosis  in  nephritis,  the  recent  work  of  Marriott  and  Rowland18 
deserves  special  mention.  They  note  that  in  the  terminal  stages  of 
nephritis  there  is  frequently  an  existing  acidosis  as  determined 
by  diminished  carbon  dioxide  tension  of  the  alveolar  air,  and  in- 
creased hydrogen-ion  concentration  of  the  blood  or  serum,  a  dim- 
inution of  the  alkali  reserve  and  of  the  oxygen  combining  power  of 
the  hemoglobin.  They  state  that  this  acidosis  is  not  due  to  an  ac- 
cumulation of  the  acetone  bodies  as  they  do  not  appear  in  the  urine 
and  they  are  not  increased  in  the  blood.  That  it  is  not  due  to  the 
presence  of  lactic  acid  seems  to  be  proved  by  the  work  of  Lewis, 
Ryffel  and  others,19  who  showed  that  lactic  acid  is  not  increased  in 
the  blood  in  this  kind  of  acidosis.  Henderson  and  Palmer20  showed 
a  diminished  ammonia  excretion  in  severe  nephritis.  An  expla- 
nation for  this  acidosis  of  severe  nephritis  is  the  fact  that  the  kid- 
neys may  be  failing  to  excrete  the  acid  substances  which  are  or- 
dinarily formed  there.  The  regulation  of  the  acid  base  equilib- 
rium of  the  body  is  largely  brought  about  by  the  ability  of  the 
kidney  to  excrete  acid  phosphate.  In  order  to  demonstrate 
whether  or  not  this  is  true  and  whether  or  not  in  severe  nephritis 
there  is  a  consequent  accumulation  of  inorganic  phosphates  in 
the  blood,  Marriott,  Haessler,  and  Howland21  worked  out  a  sim- 


"Marriott  and  Howland:     Arch.   Int.   Med.,   Nov.   15,   1916,  vol.  xviii,  No.    5,  p.   708. 

MLewis,  Ryffel,  and  others:     Heart,  1913,  vol.  v,  p.  45. 

2°Henderson  and  Palmer:  Jour.  Biol.  Chem.,  1915,  vol.  xxi,  p.  37;  Arch.  Int.  Med., 
1915,  vol.  xvi,  p.  109. 

"Howland,  Haessler,  and  Marriott:  The  Use  of  a  New  Reagent  for  Microcolorimetric 
Analysis  as  Applied  to  the  Determination  of  Calcium  and  of  Inorganic  Phosphates  in  the 
Blood  Serum,  Jour.  Biol.  Chem.,  March,  1916,  proc.  xviii,  vol.  xxiv,  No.  3. 


186  BLOOD   AND   URINE    CHEMISTRY 

pie  method  to  determine  these  inorganic  phosphates'  in  a  small 
quantity  of  serum. 

This  method  is  based  upon  the  fact  that  the  red  color  of  a  solution 
of  ferric  thiocyanate  is  discharged  by  certain  substances,  among 
which  are  oxalates  and  phosphates.  Calcium  is  precipitated  as  the 
oxalate,  dissolved  in  acid,  added  to  a  standard  solution  of  ferric 
thiocyanate  and  made  up  to  a  definite  volume.  The  color  of  the 
resulting  solution  is  compared  with  that  of  a  solution  containing 
known  amounts  of  calcium  oxalate  and  ferric  thiocyanate.  The 
phosphates  are  precipitated  as  a  magnesium  and  ammonium 
phosphate.  The  precipitate  is  dissolved  and  color  comparisons 
are  made  as  above. 

In  a. personal  communication,  Marriott  and  Haessler  give  more 
elaborate  details  on  this  micro-determination  of  inorganic  phos- 
phates in  the  serum,  as  follows : 

"Dilute  1  c.c.  of  clear,  nonhemolyzed  serum  with  5  or  10  c.c. 
of  water.  Add  two  drops  of  N/10  HC1  and  1  c.c.  of  'magnesium 
mixture.'*  Kun  in,  drop  by  drop,  with  stirring,  2  c.c.  of  10% 
ammonia  (1  volume  concentrated  ammonia  to  9  of  water) — allow 
to  stand  over  night  at  room  temperature  in  order  to  complete  pre- 
cipitation. Filter  off  precipitate  on  a  10  c.c.  Grooch  crucible,  the 
mat  being  prepared  as  follows:  A  small  disc  of  filter  paper  is 
first  placed  in  the  bottom,  asbestos  soup  is  poured  on  to  make  a 
fairly  thick  mat, — another  disc  of  filter  paper  is  laid  on  and 
then  a  little  more  asbestos,  finally  a  suspension  of  purified  barium 
sulphate  is  poured  on.  This  latter  serves  to  make  evident  any 
leaks  in  the  crucible  and  also  to  close  the  pores. 

"Wash  the  precipitate  four  times,  each  time  with  5  c.c.  of  the 
10%  ammonia,— then  twice  with  10  c.c.  portions  of  95%  alcohol 
and  finally  twice  with  10  c.c.  portions  of  ether.  The  crucible  is 
put  back  in  the  beaker  and  allowed  to  dry  over  night  at  room 
temperature  or  for  an  hour  in  an  air  bath  at  50°.  The  washing 
with  alcohol  and  ether  is  to  remove  lipoids. 

"Ten  c.c.  of  N/100  HC1  is  run  into  the  crucible  and  the  beaker 

'Magnesium  mixture  is  prepared  as  follows: 

Magnesium    chloride    sticks,  10  gm. 

Ammonium    chloride,  5  gm. 

Dissolve    in    250    c.c.    of    water    and    add    am- 
monium  hydrate    (cone.),  10  c.c. 

Allow  to  stand  over  night  to  allow  impurities  to  settle.   Filter,  neu- 
tralize with  hydrochloric  acid,  and  make  up  to  500  c.c. 


ACIDOSIS  187 

covered  tightly  with  a  piece  of  rubber  tissue  secured  with  a  rub- 
ber band  and  allowed  to  stand  several  hours  to  complete  the  solu- 
tion of  the  precipitate.  The  asbestos  is  then  thoroughly  stirred 
up  in  the  acid  and  the  suspension  poured  off  into  a  small  tube 
and  centrifuged.  An  aliquot  portion  (usually  6  c.c.)  of  the  clear 
supernatant  liquid  is  pipetted  off  and  used  for  the  determination. 

' '  COLORIMETRIC  COMPARISON. — Ammonium  TMocyanate  Solution 
(3  grams  to  1000  c.c.  ferric  chloride  solution). — Weigh  out  3 
grams  of  ferric  chloride  with  its  contained  water  of  crystalliza- 
tion. Dissolve  in  water  and  add  just  sufficient  HC1  to  make  a 
clear  solution  and  make  up  to  1000  c.c.  Just  before  use,  these 
solutions  are  mixed  by  taking  5  c.c.  each  and  making  up  to  from 
35  to  50  c.c.  with  distilled  water,  this  solution  being  used  more 
dilute  for  serum  containing  small  amounts  of  phosphate.  Ac- 
curately measured  2  c.c.  portions  of  the  iron  thiocyanate  solution 
thus  prepared  are  measured  into  10  c.c.  volumetric  flasks;  the 
aliquot  portions  of  the  phosphate  solution  are  added  in  the  flask 
and  the  liquid  made  up  to  the  mark  with  N/100  HC1.  Known 
amounts  of  a  standard  solution  of  magnesium  ammonium  phos- 
phate in  N/100  HC1  are  added  to  other  10  c.c.  flasks  containing 
thiocyanate  and  made  up  to  the  mark  with  N/100  HC1.  Color 
comparisons  are  made  in  small  glass  tubes  approximately  120 
mm.  long  by  10  mm.,  internal  diameter.  The  tubes  are  filled  to 
the  same  height  and  compared  by  looking  through  them  length- 
wise against  a  white  surface.  The  colors  do  not  change  within 
an  hour's  time. 

"Standard  Magnesium  Ammonium  Phosphate  Solution. — Dis- 
solve .1584  gm.  of  air  dried  MgNH4Po4 .  6H20  in  100  c.c.  of  N/10 
hydrochloric  acid  and  dilute  to  1  liter  with  water.  Of  this  solu- 
tion, 1  c.c.  .02  mgm.  phosphorus. 

"Additional  notes  and  cautions  on  the  determinations  of  cal- 
cium and  inorganic  phosphate  are  given  as  follows: 

"CALCIUM  METHOD. — In  the  ashing  of  the  blood  by  nitric  acid 
a  certain  amount  of  difficultly  soluble  calcium  sulphate  is  formed. 
This  is  especially  insoluble  if  the  liquid  is  allowed  to  go  to  dry- 
ness.  In  all  cases,  it  is  advisable  after  the  nitric  acid  has  evap- 
orated to  add  distilled  water  to  the  flask  and  to  heat  on  a  sand 


188  BLOOD   AND   URINE    CHEMISTRY 

bath  just  below  boiling  for  one  hour  or  more,  in  order  to  com- 
pletely bring  the  calcium  into  solution. 

"By  '20%'  sodium  acetate  is  meant  20%  of  anhydrous  sodium 
acetate.  If  the  crystalline  salt  is  used  the  solution  should  be 
35%. 

"The  beakers  used  in  the  method  should  be  of  the  tall,  narrow 
type  rather  than  of  the  broad  form  as  in  this  way  the  solution 
of  the  precipitate  seems  to  be  more  complete.  Instead  of  centri- 
fuging  the  asbestos  suspension  before  removing  an  aliquot  por- 
tion, filtration  may  be  resorted  to.  Results  obtained  are  the  same. 

"In  the  colorimetric  comparison  of  calcium  and  of  phosphates, 
instead  of  using  10  c.c.  volumetric  flasks,  it  is  convenient  to  have 
a  set  of  small  flat-bottomed  Nessler  tubes,  approximately  120  mm. 
long,  10  mm.  internal  diameter,  these  tubes  being  of  exactly  the 
same  size  and  with  a  graduation  at  the  10  c.c.  mark.  The  solu- 
tions may  be  made  up  in  these  tubes  and  mixed  by  inverting.  In 
that  way  the  volumetric  flasks  may  be  dispensed  with. 

"  PHOSPHATE  "  METHOD. — In  making  up  the  standard  solutions, 
it  is  to  be  borne  in  mind  that  MgNH4Po4 .  6H2O  loses  water  of 
crystallization  if  heated,  and,  therefore,  must  be  dried  at  room 
temperature.  Commercial  preparations  of  this  salt  are  unreliable. 
It  should  be  prepared  by  precipitation  and  dried  as  directed."' 

By  this  method  they  determined  the  inorganic  phosphates  in 
the  serum  of  a  series  of  normal  adults  and  older  children  and 
then  of  patients  with  nephritis,  both  with  and  without  acidosis. 
The  normal  figure  expressed  in  terms  of  phosphorus  varied  from 
1  to  3.5  mgms.  per  100  c.c.  of  blood.  In  the  great  majority  of 
normals  the  amount  was  less  than  2  mgms.  They  also  determined 
the  inorganic  phosphate  in  the  serum  of  patients  with  acidosis 
occurring  in  the  course  of  nephritis  and  in  every  instance  they 
found  an  increase  in  the  phosphorus  to  many  times  the  normal, 
that  is,  an  increase  up  to  23  mgms.  per  100  c.c.  of  blood.  They  be- 
lieve that  the  retention  of  the  acid  phosphate  (for  approximately 
90  per  cent  of  the  phosphate  in  an  average  urine  is  acid  phos- 
phate) would  seem  to  be  sufficient  to  account  for  the  degree  of 
acidosis  observed.  They  do  not  claim  that  this  is  the  sole  fac- 
tor in  this  acidosis  of  nephritis,  but  they  point  to  the  fact  that  the 
retention  of  acid  phosphate  in  nephritis  is  not  part  of  a  general 


ACIDOSIS  189 

salt  retention;  that  it  seems  to  be  due  to  a  certain  "specificity" 
of  retention  because  there  was  no  corresponding  increase  of  so- 
dium chloride  with  the  increase  in  acid  phosphate.  It  was  not 
proportional  to  the  total  nitrogen  and  the  urea  retention  in 
these  cases.  In  other  words  the  phosphate  retention  \vas  not 
a  result  of  the  acidosis  per  se,  for  these  writers  failed  to  find  a 
similar  increase  in  the  inorganic  phosphate  in  that  form  of  acid- 
osis seen  in  diabetics.  They  believe  that  the  phosphate  is  due 
to  some  disturbance  in  the  specific  function  of  the  kidney  and 
not  to  increased  phosphate  production  in  the  body  or  increased 
absorption  from  the  intestinal  canal,  because  the  urinary  output 
of  phosphate  is  not  increased  and  may  even  be  decreased.  They 
failed  to  reduce  this  phosphate  retention  by  the  administration 
of  alkali  and  even  demonstrated  an  increase  of  the  substance 
under  sodium  bicarbonate  administration. 

They  also  found  in  these  cases  a  marked  reduction  in  the  cal- 
cium of  the  serum,  in  one  case  as  low  as  1.5  mgms.  per  100  c.c. 
of  serum  as  compared  with  the  normal  of  10  to  11  mgms.  The 
low  calcium  is  to  be  referred  to  an  excess  of  phosphates  in  the 
serum,  as  already  detailed.  The  administration  of  phosphates 
causes  an  increased  elimination  of  calcium  through  the  feces,  and 
the  converse  is  also  true ;  the  administration  of  calcium  leads  to 
an  increased  elimination  of  phosphate,  also  by  the  intestines. 
This  fact,  according  to  these  investigators,  may  offer  a  suggestion 
for  rational  therapeutic  procedure. 

At  the  May,  1916,  meeting  of  the  Association  of  American  Phy- 
sicians, a  very  excellent  summing  up  of  the  entire  question  of 
acidosis  was  gone  into  by  the  leaders  on  this  question ;  namely,  L. 
J.  Henderson,  of  Boston,  John  Howland,  of  Baltimore,  R.  T. 
Woodyatt,  of  Chicago,  C.  Frothingham,  of  Boston,  L.  G.  Rown- 
tree,  of  Minneapolis,  Yandell  Henderson,  of  New  Haven,  and 
Donald  Van  Slyke,  of  New  York.  It  recapitulates  most  of  what  we 
have  discussed,  so  we  will  abstract  it  here.  In  this  symposium  on 
acidosis,22  L.  J.  Henderson,  speaking  on  the  subject  of  the  biochem- 
istry of  acidosis,  said  that  like  heat  equilibrium,  the  equilibrium  be- 
tween acids  and  bases  was  essential  to  life.  Fluctuations  in  equilib- 
rium occurred,  but  normally  the  limits  of  fluctuation  were  narrow. 


"New  York  Med.  Jour.,  Dec.  2,  1916,  p.  1119. 


190  BLOOD   AND   URINE    CHEMISTRY 

Wider  fluctuations  occurred  pathologically,  but  the  acid  base  fluc- 
tuations did  not  as  a  rule  involve  changes  in  the  hydrogen-ion 
concentration.  Acidosis  was  defined  as  any  disturbance  of  the 
acid-basic  equilibrium  whereby  the  power  to  resist  acids  in  the 
body  was  lost.  It  is  now  possible  to  say  that  the  main  change  in 
acidosis  is  the  loss  of  blood  bicarbonates.  The  bicarbonates  were 
to  be  regarded  as  the  third  constituent  of  the  blood;  reckoning 
water  first,  salt  second,  and  bicarbonate  third.  This  third  con- 
stituent is  specially  subject  to  fluctuations,  owing  to  the  constant 
physicochemical  interchanges  between  blood  and  respired  air ;  and 
since  hydrogen-ion  concentration  is  proportional  to  the  reactions 
between  bicarbonates  and  free  carbon  dioxide,  the  ratio  of  free 
carbon  dioxide  and  bicarbonates  is  kept  fairly  constant  by  the 
mechanism  of  ventilation;  hence,  hydrogen-ion  concentration  is 
now  regarded  as  the  hormone  of  respiration.  The  maintenance 
of  the  acid  basic  equilibrium  becomes  more  complicated  in  patho- 
logical states,  and  is  always  related  to,  and  dependent  on  the  gen- 
eral metabolism  of  the  body.  Beneath  all  metabolism  is  a  constant 
diminution  of  blood  carbonates;  unless  repaired,  this  leads  to 
acidosis.  The  carbon  dioxide  tension  of  alveolar  air  and  of  the 
blood,  together  with  the  measure  of  alkali  ingestion  are  the  meas- 
ures of  acidosis.  Neither  ammonia  concentration  nor  urinary 
findings  are  safe  guides.  Attempts  to  explain  general  pathological 
states  on  the  basis  of  hydrogen-ion  concentration  or  acidosis  are 
not  justified.  Any  attempt  to  treat  a  disease  like  nephritis  by  the 
indiscriminate  administration  of  large  amounts  of  alkali  is  mal- 
practice. Small  amounts  of  alkali,  given  over  a  long  time,  are 
allowable,  and  when  so  given,  acidosis  is  impossible. 

Howland,  speaking  on  "Acidosis  in  Infants  and  Children,"  re- 
peated some  of  the  facts  already  credited  to  him  in  the  preceding 
pages.  He  noted  that  acidosis  in  children  is  a  dangerous,  but 
not  often,  an  acute,  self-limited  disease.  It  is  not  merely  an 
acetonuria,  but  is  dependent  upon  a  loss  of  the  acid  basic  equilib- 
rium of  the  blood.  Hyperpnea,  as  noted  before,  is  the  clinical 
sign;  laboratory  tests  are  the  indices,  these  being  carbon  dioxide 
tension  of  alveolar  air,  hydrogen-ion  concentration  of  blood,  and 
alkali  reserve  of  blood.  The  natural  low  level  of  carbon  dioxide 
tension  and  low  hydrogen-ion  concentration  in  the  young  ex- 


ACIDOSIS  191 

plains  the  susceptibility  to  acidosis.  Onset  of  acidosis  is  marked 
by  hyperpnea;  coma  soon  ensues;  the  alkali  reserve  might  be  re- 
stored, but  unless  this  occurs  quickly,  death  follows.  When  acid 
phosphates  are  found  in  excess  (five  to  fifteen  times  the  normal) 
in  the  blood,  and  this  condition  continued  long  enough,  it  robs 
the  body  of  its  bases.  Restoration  of  bases  does  not  always  stop 
the  accumulation  of  acid  phosphates.  Acidosis  is  seen  in  many 
diseases  of  infancy  and  childhood  and  should  always  be  kept  in 
mind;  its  early,  rational  treatment  may  be  the  means  of  saving 
life. 

The  next  paper  in  the  symposium  was  that  of  R.  T.  Woodyatt  on 
"Acidosis  in  Diabetes."  He  explained  that  the  occurrence  of 
acidosis  in  diabetes  depended  on  the  definition  of  the  difference 
between  the  diabetic  and  the  normal  individual.  The  proportional- 
ity between  glucose  utilization  and  wastage  depended  upon  the 
rate  of  intake.  It  may  be  said  that  with  a  rate  of  glucose  in- 
take high  enough,  the  normal  subject  became  diabetic;  with  the 
intake  low  enough,  the  diabetic  acts  like  the  normal  individual. 
The  difference  was  in  the  wastage.  The  occurrence  of  acidosis 
in  diabetes  depends  upon  this;  for  it  has  been  found  that  one 
molecule  of  carbohydrate  must  be  burnt  to  care  for  three  mole- 
cules of  higher  fatty  acids;  if  this  ratio  can  be  maintained,  the 
body  "smoked"  with  unburnt  fats,  acetone,  beta-oxybutyric  acid 
and  diacetic  acid  appear  in  the  urine.  In  diabetics  the  absolute 
rate  of  carbohydrate  utilization  is  low  and  it  is  necessary  to  bend 
down  the  rates  of  protein  and  fat  metabolism  to  meet  that  of  the 
carbohydrates.  Thus  the  application  of  rest,  warmth  and  fasting 
in  diabetes  is  rational.  Acidosis  in  diabetes  may  be  accounted  for 
always  in  the  way  described,  except  in  certain  cases ;  e.g.,  its  occur- 
rence in  the  course  of  septic  processes;  such  may  be  called  ac- 
cidental rather  than  diabetic  acidoses. 

Referring  to  "Acidosis  in  Acute  and  Chronic  Diseases,"  Froth- 
ingham  said  that  the  finding  of  acidosis  in  diseased  states  other 
than  diabetes  led  to  a  study  of  carbon  dioxide  tension  of  alveolar 
air,  hydrogen-ion  concentration  in  blood,  acetone  and  ammonia 
nitrogen  output  in  urine,  and  soda  utilization  in  a  large  and  di- 
versified series  of  cases. 

The  very  key-note  of  the  discussion  on  acidosis  was  furnished 


192  BLOOD  AND  URINE  CHEMISTRY 

by  Dr.  Yandell  Henderson,  who  emphasized  the  fact  that  in  a 
discussion  on  acidosis,  one  writer  speaks  about  one  thing  and  an- 
other about  an  entirely  different  aspect  of  the  question.  There  is 
need  here,  as  in  other  medical  discussions,  of  a  clear  cut  nomencla- 
ture. It  goes  without  saying  that  the  acidosis  of  former  days  is 
not  the  acidosis  of  today.  The  acidosis  of  nephritis  is  not  the 
acidosis  of  diabetes.  Henderson  urged  that  it  might  be  better 
to  speak  in  one  case  of  a  ketonuria  and  in  another  of  low  carbon 
dioxide  states,  and  so  on.  In  1911  he  was  a  member  of  Haldane's 
Pike's  Peak  expedition,  and  all  of  the  party  had  acidosis  when 
a  sufficient  altitude  was  reached,  if  the  carbon  dioxide  tension  was 
taken  as  an  index.  Henderson  was  very  skeptical  of  the  hurtful 
effects  of  acidosis,  for  he  had  seen  no  figures  which  indicated  a 
more  severe  acidosis  than  he  persistently  had  himself  on  Pike's 
Peak  when  feeling  particularly  well.  The  description  given  by 
Dr.  Lawrence  Henderson  was  on  the  basis  of  sea  level  data.  But 
on  going  above  sea  level  acidosis  increased  with  the  altitude ;  in  a 
caisson,  acidosis  diminished.  Miss  Fitzgerald,  of  the  Haldane 
expedition,  had  shown  this  as  a  result  of  hundreds  of  observa- 
tions made  by  her  at  various  altitudes.  The  net  result  of  her 
work  was  that  one  could  determine  the  altitude  of  any  commun- 
ity by  the  measure  of  the  carbon  dioxide  tension  of  the  alveolar 
air  of  the  inhabitants,  or  in  other  words,  by  their  acidosis.  It 
seemed,  therefore,  to  Henderson,  much  safer  to  keep  in  mind  the 
facts ;  from  the  urinary  standpoint,  acetonuria  may  be  found ; 
from  the  respiratory  standpoint,  variations  in  carbon  dioxide  ten- 
sion, or  volume  of  ventilation  might  be  measured ;  from  the  point 
of  view  of  the  blood,  disturbances  of  hydrogen-ion  concentration 
might  be  noted ;  and  other  measures  of  the  body 's  alkali  acid  bal- 
ance might  be  made.  But  if  all  these  measures  were  to  be  accepted 
as  measures  of  acidosis,  conditions  of  acidosis  would  be  met  with 
in  which  the  acidosis  was  not  a  condition  of  acid  blood  at  all, 
because  the  hydrogen-ion  concentration  of  the  blood  might  still 
be  normal.  It  is  therefore  necessary  to  formulate  and  keep  clear- 
ly in  mind  just  what  in  the  future  is  to  be  known  as  acidosis. 

Van  Slyke,  concluding  this  very  interesting  discussion  on  acid- 
osis, called  attention  to  the  fact  that  he  and  his  co-workers  had  been 
much  interested  in  the  relations  between  the  kidney,  lung,  and 
blood  functions  in  acidosis  and  their  observations  had  led  them 


ACIDOSIS  193 

to  conclude  that  the  phenomena  arising  in  the  various  systems 
were  the  corollaries  one  to  another.  He  had  been  struck  by  the 
beautiful  concord  between  the  clinical  and  the  chemical  facts,  and 
the  theoretical  considerations  advanced  originally  by  Lawrence 
Henderson.  Van  Slyke  thought  that  acidosis  was  a  loss  of  the 
normal  relationship  between  acids  and  the  bicarbonates  of  the 
blood.  He  also  believed  in  Rowntree's  classification  of  compen- 
sated acidosis  and  true  acidosis  on  the  basis  of  undisturbed  hydro- 
gen-ion concentration  respectively.  He  believed  that  the  reduc- 
tion of  carbon  dioxide  tension  of  alveolar  air  is  only  an  indirect 
measure  of  hydrogen-ion  concentration  of  the  blood  and  cannot 
be  regarded  as  synonymous  with  acidosis.  It  is  an  exact  measure 
of  the  hydrogen-ion  state  of  the  blood  only  when  the  lungs  are 
functioning  normally  and  under  fixed  conditions  of  temperature 
and  atmosphere.  The  same  may  be  said  of  the  urinary  findings: 
certain  urinary  changes  are  recognizable  and  acceptable  as  indi- 
rect evidences  of  acidosis :  but  they  are  not  synonymous  with 
acidosis,  and  depend  upon  renal  integrity  and  other  factors  for 
constancy. 


CHAPTER  XXVII. 
BLOOD  CHANGES  IN  GOUT. 

Among  other  conditions  in  which  blood  chemistry  has  played 
a  role  in  differential  diagnosis,  might  be  mentioned  gout  and 
rheumatism.  This  disease  which  was  most  accurately  described 
by  Sydenham  (London,  1763)  is  a  peculiar  condition  about  the 
etiology  of  which  there  still  prevails  much  confusion.  However, 
it  may  perhaps  conservatively  be  stated  at  this  time  that  it  is  a 
chronic  disorder  of  metabolism  in  which  there  is  an  undue  ac- 
cumulation of  uric  acid  in  the  blood  as  a  result  of  a  disturbance 
in  the  endogenous  and  the  exogenous  uric  acid  formation.  Gar- 
rod1  as  long  ago  as  1848  contended  that  in  gout  we  have  an  excess 
of  uric  acid  in  the  blood  due  to  increased  formation  and  decreased 
elimination.  •  Present-day  methods  of  blood  chemical  analyses 
seem  to  prove  that  he  was  correct  in  his  views,  i.  e.,  that  in  gout 
we  have  an  undue  accumulation  of  uric  acid  over  the  normal 
figure  (1-3.0  mgms.  per  100  c.c.  of  blood),  whereas  in  rheumatism 
there  is  no  such  accumulation,  the  figure  remaining  around  1  to 
3.0  mgms.  Without  going  too  deeply  into  the  theories  on  the 
cause  of  this  disturbance  of  metabolism,  we.  might  simply  state 
that  according  to  Brugsch  and  Schittenheim,2  gout  results  from 
metabolic  disturbances  due  to  changes  in  the  conversion  of  the 
purin  bases.  Folin  and  Denis3  showed  that  the  amount  of  uric 
acid  in  the  blood  under  normal  conditions,  using  their  colori- 
metric  methods,  varied  from  0.7  to  3.7  mgms.  per  100  c.c.  of 
blood.  Adler  and  Ragle'4  reported,  in  156  patients,  a  variation 
in  uric  acid  from  0.7  to  4.5  mgms.  per  100  grams  of  blood.  These 
cases  were  taken  at  random  from  hospital  cases  and  included 
conditions  such  as  chronic  interstitial  nephritis  in-  which  there 
might  be  expected  some  increase  in  the  normal  amount  of  uric 


'Garrod,  A.  B. :     Med.   Clin.,   1848,  vol.  xxxi,  p.  83;  and  Treatise  on  Gout  and  Rheu- 
matic Gout,  1876. 

'Brugsch:     Gicht.  Spec.  Path.  u.  Ther.,  1913,  Lieferung,  I-IV,  Wien  u.  Berlin. 
Brugsch  and   Schittenheim:     Gicht.     Jena,   1910. 
"Folin  and  Denis:     Jour.  Biol.   Chem.,  1913,  vol.  xiv,  p.  82. 
4Adler  and  Ragle:     Boston  Med.  and  Surg.  Jour.,   1914,  vol.  clxxi,  p.   769. 


BLOOD    CHANGES    IN    GOUT  195 

acid.  It  was  formerly  supposed  that  uric  acid  could  not  be  found 
in  the  blood  of  normal  persons  who  were  placed  upon  a  purin- 
free  diet.  Its  constant  appearance  with  the  patient  on  this  diet 
was  regarded  in  the  nature  of  things  as  a  test  meal  method  of 
proving  the  existence  of  gout.  That  this  was  entirely  erroneous 
has  been  proved  time  and  again.  For  instance,  McLester,5  using 
the  method  of  Folin,  found  uric  acid  in  the  blood  of  fifteen  nor- 
mal individuals  who  had  been  on  a  purin-free  diet  for  at  least 
three  days,  in  amounts  varying  from  0.5  to  2.9  mgms.  per  100 
c.c.  of  blood.  Pratt6  showed  the  remarkable  changes  of  uric  acid 
in  gout.  He  reported  in  1913  eleven  cases  of  typical  gout  in 
which  the  uric  acid  in  the  blood  had  been  determined  by  the 
method  of  Folin  and  Denis  in  Folin 's  laboratory.  In  a  subsequent 
paper  he  reports7  the  number  of  cases  studied  as  sixteen.  He  in- 
cludes only  cases  in  which  tophi  were  found,  or  in  which  a  his- 
tory of  characteristic  attacks  of  acute  gout  was  obtained  or  in 
which  typical  symptoms  developed  while  under  observation. 
Pratt 's  findings  are  quite  interesting  and  deserve  special  men- 
tion. The  average  amount  of  uric  acid  irrespective  of  the  diet  or 
the  condition  of  the  patient  at  the  time  of  the  examination  was 
3.7  mgms.  Three  of  the  patients  seen  during  the  attacks  were 
on  an  ordinary  mixed  diet.  They  had  4.5  mgms.,  4.8  mgms.,  and 
5.7  mgms.  of  uric  acid.  In  the  blood  of  two  other  patients  examined 
during  an  attack  while  on  a  purin-free  diet,  the  uric  acid  in  four 
determinations  ranged  from  2.4  to  5.1  mgms.,  with  an  average 
amount  of  3.6  mgms.  None  of  these  patients  were  taking  atophan. 
Seven  patients  were  examined  at  a  time  when  they  were  free 
from  symptoms  of  gout  and  when  they  were  on  a  mixed  diet.  Their 
blood  contained  from  3.1  to  5.5  mgms.  The  average  was  4.3  mgms. 
In  the  blood  of  six  patients  examined  when  they  were  on  a  purin- 
free  diet  and  having  no  acute  symptoms,  Pratt  found  uric  acid 
values  from  1.6  to  7.2  mgms.,  an  average  of  3  mgms.  These 'figures 
showed  that  in  the  cases  studied  there  was  more  uric  acid  in  the 
blood  when  on  a  mixed  diet  both  in  the  interval  and  during  attacks 
than  when  on  a  purin-free  diet.  In  all,  twelve  examinations  were 
made  when  a  mixed  diet  was  taken  during  attacks  as  well  as  in 


5McLester:     Arch.  Int.   Med.,  1913,  vol.  xii,  p.   737. 

6Pratt:     Tr.  Am.  Assn.   Physicians,   1913,  vol.  xxviii,  p.   387. 

7Pratt:     Am.  Jour.  Med.   Sc.,   1916,  vol.  cli,  No.   1,  p.  92. 


196  BLOOD   AND   URINE    CHEMISTRY 

the  intervals,  and  the  average  amount  of  uric  acid  was  4.3  mgms. 
The  general  conclusion  from  these  figures  is  that  in  gout  there 
is  always  a  hyperuricemia.  Thirty-eight  examinations  made  on 
sixteen  cases  of  gout  showed  an  average  amount  of  uric  acid 
of  3.7  mgms.  per  100  c.c.  of  blood.  It  is  generally  believed  that 
there  is  more  uric  acid  in  the  blood  during  an  acute  attack  than 
in  the  intervals,  but  this  is  not  always  true.  Pratt 's  figures  show, 
and  other  investigators  corroborate  them,  that  while  in  gout  there 
is  a  relatively  large  amount  of  uric  acid,  the  diagnosis  of  gout 
cannot  be  based  absolutely  upon  a  single  blood  test:  there  is  a 
high  concentration  found  at  times  in  other  joint  conditions.  But 
it  must  be  remembered  that  in  gout  the  condition  of  hyperuri- 
cemia is  long-continued,  while  in  the  other  joint  conditions  it  is 
transitory.  The  obvious  procedure,  therefore,  is  to  follow  one 
examination  up  with  others  at  interrupted  intervals  of  time.  For 
instance,  one  of  Pratt 's  cases  of  infectious  arthritis  without  any 
of  the  clinical  features  of  gout,  showed  at  the  time  of  the  first 
examination  7.6  mgms.  of  uric  acid.  Seven  months  later  the 
blood  was  again  analyzed  and  only  0.8  mgms.  found,  although 
the  patient  was  then  on  a  diet  rich  in  purins.  Other  cases  have 
shown  the  necessity  of  repeated  blood  examinations. 

It  seems  that  there  is  no  relationship  between  the  amount  of 
uric  acid  retained  in  gout  and  the  severity  of  the  disease.  Again, 
the  age  of  the  patient  has  no  bearing  upon  this  question.  Atten- 
tion must  also  be  called  to  the  fact  that  the  retention  of  uric 
acid  is  in  no  way  to  be  determined  by  a  diminution  in  the  output 
of  uric  acid  in  the  urine.  Vogt,8  Reach,9  and  others  have  at- 
tempted to  show  that  in  gout  the  excretion  of  exogenous  purin 
is  diminished.  Magnus-Levy,10  however,  has  disproved  this  com- 
pletely, and  Pratt 's11  figures  show  that  a  marked  increase  and 
retention  of  uric  acid  in  the  blood  may  result  from  the  ingestion 
of  purin  bases  even  when  no  evidence  of  retention  is  found  on 
examination  of  the  urine.  A  number  of  experimental  test  meals 
given  for  the  purpose  of  determination  of  whether  or  not  the  giv- 
ing of  purin-rich  diets  can  increase  the  uric  acid  in  the  blood 
of  healthy  people  shows  that  they  cannot  do  so.  It  has  been 

8Vogt:     Deutsch.  Arch.  f.  klin.  Med.,   1901,  vol.  Ixxi,  p.  21. 
9Reach.     Munchen.  med.  Wchnschr.,   1902,  vol.  xlix,  p.  215. 
"Magnus- Levy:      Deutsch.    med.    Wchnschr.,    1911,  vol.   xxvii,   p.    778. 
"Pratt:     Am.  Jour.  Med.   Sc.,  1916,  vol.  cli,  No.   1,  p.  92. 


BLOOD  CHANGES  IN  GOUT  197 

clearly  proved  that  the  uric  acid  derived  from  exogenous  purin 
does  not  accumulate  in  the  blood  unless  there  is  a  disturbance  in 
the  uric  acid  metabolism. 

We  have  abundant  analytical  evidence  to  prove,  therefore,  that 
in  gout  there  is  increase  in  the  uric  acid  concentration  in  blood 
without  any  increase  in  the  other  nonprotein  nitrogenous  constitu- 
ents. Daniels  and  McCrudden  are  two  observers  who  have  reported 
several  cases  of  gout  in  women  without  any  increase  in  uric  acid  in 
the  blood.  No  one  else  has  found  normal  figures.  On  the  con- 
trary, Fine,  who  has  contributed  a  great  deal  to  the  literature 
on  uric  acid  values  in  gout  and  other  conditions,  states  that  he 
has  never  seen  normal  uric  acid  in  blood  in  gout.  It  would  seem 
that  in  the  estimation  of  the  amount  of  uric  acid  in  the  blood  we 
have  an  excellent  method  of  differentiating  gout  from  rheuma- 
tism and  other  joint  affairs.  This  is  clearly  evident.  It  must 
be  remembered,  however,  that  the  increase  in  uric  acid  alone 
without  any  increase  of  urea  nitrogen  and  creatinine,  may  occur 
in  early  chronic  interstitial  nephritis.  In  a  recent  communica- 
tion, entitled:  "The  Relation  of  Gout  to  Nephritis  as  Shown 
by  the  Uric  Acid  in  the  Blood,"  Fine,12  goes  thoroughly  into  this 
question.  He  states  that  while  uric  acid  concentrations  up  to  4 
to  9  mgms.  in  blood  are  found  in  gout,  these  accumulations  are 
not  infallible  signs  of  gout.  Indeed,  Garrod,13  von  Jaksch,14 
and  von  Noorden15  pointed  this  out  in  connection  with  the  reten- 
tion of  uric  acid  as  well  as  urea,  but,  of  course,  their  observations 
were  purely  clinical.  Owing  to  the  fact  that  in  early  interstitial 
nephritis  there  is  only  an  undue  retention  of  uric  acid  in  the  blood, 
it  is  necessary  to  exclude  this  condition  before  adopting  the  diag- 
nosis of  gout.  Fine  states  that  there  may  be  no  undue  accumula- 
tion of  urea  nitrogen  and  creatinine  in  early  interstitial  nephritis, 
uric  acid  values  alone  showing  an  abnormal  figure  over  2.5  mgms. 
He  showed  in  collaboration  with  Myers  and  Lough16  very  plainly 
that  in  early  interstitial  nephritis,  there  is  first  an  accumulation  of 
uric  acid ;  secondly  an  accumulation  of  urea,  and,  finally,  an  ac- 


"Fine:     Jour.  Am.  Med.  Assn.,  1916,  vol.  Ixvi,  No.  26. 

"Garrod,  A.   B. :     Med.   Clin.,   1848,  vol.   xxxi,  p.   83;  and  Treatise  on  Gout  and  Rheu- 
matic Gout,  1876. 


Dn  Jaksch:     Zentralbl.  f.  inn.  Med.,  1896,  vol.  xvii,  p.  545. 
HI  Nc 


15von  Noorden:     Metabolism   and   Practical   Medicine,   1907,  vol.   iii,   p.   29;   Ibid.,    1914, 

l.  xvii,  p.  487. 

"Myers,  Fine  and  Lough:     Arch.  Int.  Med.,   1916,  pp.   570-583. 


198  BLOOD   AND   URINE    CHEMISTRY 

cumulation  of  creatinine  in  the  blood.  This  is  what  these  ob- 
servers term  their  "stair-case"  effect.  Twelve  cases  came  under 
their  observation  in  which  more  than  10  mgms.  of  uric  acid  were 
found  in  the  blood  without  any  gouty  symptoms.  In  one  case 
as  much  as  27  mgms.  were  present.  It  was  also  observed  by  them 
that  higher  uric  acid  values  were  seen  early  in  the  cases  than 
later,  although  during  the  agonal  period  there  was  a  marked 
increase  coincident  with  that  of  creatinine.  Folin  and  Denis17  re- 
marked on  the  fact  that  in  the  severest  cases  of  uremia  there  was 
only  a  slight  increase  in  the  blood  ammonia  and  that  it  was  like- 
wise only  these  cases  in  which  a  marked  retention  of  creatinine 
occurred.  They  concluded  from  this  that  the  human  kidney  re- 
moves the  creatinine  from  the  blood  with  remarkable  ease  and 
certainty.  The  completeness  of  the  creatinine  excretion,  is,  in 
fact,  they  further  state,  exceeded  only  by  the  still  more  complete 
removal  of  the  ammonium  salts. 

Myers,  Fine,  and  Lough18  give  in  tabular  form  some  interesting 
data  showing  in  a  series  of  twenty-six  cases  studied,  a  decided 
increase  in  the  concentration  of  the  uric  acid  alone  without  any 
corresponding  increase  in  urea  nitrogen  or  creatinine.  Some  of 
these  cases  showed  symptoms  which  in  general  are  characteristic 
of  early  interstitial  nephritis.  In  other  cases,  although  the  nephri- 
tis was  not  the  predominant  clinical  condition,  it  would  appear 
that  the  systemic  disturbances  resulting  from,  or  associated  with, 
a  variety  of  conditions,  such  as  tuberculosis,  typhoid  fever,  pneu- 
monia, carcinoma,  cardiac  disorders,  chronic  alcoholism,  etc.,  are 
capable  of  exerting  the  same  influence  upon  the  kidney.  It  is 
not  improbable  that  similar  factors  are  at  work  in  gout  and  the 
apparently  uncomplicated  cases  of  interstitial  nephritis.  These 
investigators  also  showed  in  tabular  form  four  cases  of  diabetes  with 
uric  acid  values  of  10.5,  6.0,  5.0,  and  7.6  mgms.  respectively  where 
there  were  similarly  normal  creatinine  values,  namely,  2.1,  2.0, 
2.3  and  4.7  mgms.,  respectively.  The  last  figure,  of  course,  is  an 
increase  in  creatinine.  In  this  case  the  patient  entered  the  hos- 
pital in  coma  and  died  several  hours  later;  the  urine  contained 
very  large  amounts  of  albumin,  acetone,  and  diacetic  acid,  and 
many  granular  casts.  These  observers  point  to  the  fact  that  their 


•^",7     ^,-»- ^Ait*A.t*i    v/c*oi;Q.         A  iicoc    UUoClVCitt    IJUlllt    LU     L 

17Folin  and  Denis:     Jour.   Biol.   Chem.,   1914,  vol.  xvii,  p.   487. 
18Myers,  Fine,  and  Lough:     Arch.  Int.  Med.,  1916,  pp.   570-583. 


BLOOD    CHANGES    IN    GOUT 


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200  BLOOD   AND   URINE    CHEMISTRY 

series  of  thirty  cases  were  apparently  suffering  with  early  inter- 
stitial nephritis,  probably  secondary  in  many  instances  to  other 
systemic  disturbances.  They  believe  that  an  increased  uric  acid 
value  alone  without  any  increase  in  urea  nitrogen  or  creatinine 
might  serve  as  an  aid  to  an  early  diagnosis.  They  also  suggest 
that  a  retention  of  uric  acid  may  be  earlier  evidence  of  renal  im- 
pairment of  an  interstitial  type  than  the  classical  tests  of  albumi- 
nuria  and  cylinduria.  In  Fine's  later  paper,19  he  gives  a  table 
(see  Table  XII,  page  199)  of  two  groups  of  cases,  the  first  com- 
posed of  five  cases  giving  the  classical  histories  of  gout,  and  the 
second  consisting  of  seven  cases  with  some  evidence  of  incipient 
nephritis,  such  as  slight  albuminuria,  cylinduria  or  diminished 
phenolsulphonphthalein  output.  These  cases  were  given  a  pur- 
in-free  diet  several  days  before  the  examinations  were  made.  The 
first  two  cases  that  he  calls  attention  to  gave  typical  histories  of 
gout,  but  also  showed  one  or  more  signs  of  nephritis  and  from 
this  standpoint  might  well  have  been  placed  in' the  second  group. 
He  points  to  the  striking  similarity  in  the  blood  pictures  in  the 
two  groups. 

There  is  truly  a  slight  increase  of  urea  nitrogen  and  creatinine 
in  group  2,  but  the  increase  is  negligible.  Fine  states  that  many 
cases  of  gout  have  been  reported20  with  blood  uric  acid  concen- 
trations as  low  or  lower  than  the  lowest  in  the  above  group  2. 
Fine  propounds  the  following  queries  as  a  result  of  these  figures : 
1.  Is  gout  merely  a  stage  in  the  development  of  interstitial  neph- 
ritis, whose  further  progress  may  be  indefinitely  delayed?  2. 
Is  early  interstitial  nephritis  merely  potential  gout,  in  which  the 
clinical  symptoms  may  or  may  not  be  eventually  in  evidence?  3. 
Is  the  uric  acid  retention  of  gout  due  to  the  specific  condition, 
gout,  or  to  a  complicating  early  interstitial  nephritis  ? 

From  these  observations  and  reports  we  can  readily  recommend 
the  advisability  of  blood  chemical  analyses  in  dealing  with  sus- 
pected cases  of  gout,  rheumatic  fever,  and  early  interstitial  neph- 
ritis. No  adequate  comprehension  of  cases  of  this  kind  can  be 
obtained  by  mere  urinary  findings  or  the  best  clinical  symptoms. 

fFine:     Jour.  Am.  Med.  Assn.,  1916,  vol.  Ixvi,  No.  26. 

°nn<Denis:   Joun  Bio1'  Chem"  1913>  vo1'  xiv>  p-  40;  atld  Arch-  Int  Med"  1915' 


CHAPTER  XXVIII. 
BLOOD  CHEMISTRY  AND  NEPHRITIS. 

It  has  already  been  noted  that  in  gout  we  have  an  alteration 
in  the  concentration  of  one  of  the  nonprotein  nitrogenous  blood 
constituents ;  namely,  uric  acid.  Attention  has  also  been  called  to 
the  fact  that  in  early  interstitial  nephritis  we  have  likewise  only 
an  accumulation  of  uric  acid.  It  will  be  necessary  in  discussing 
the  blood  figures  in  chronic  nephritis,  interstitial  or  parenchyma-' 
tous  in  variety,  to  refer  to  some  of  the  facts  of  nitrogenous  meta- 
bolism. Nonprotein  blood  constituents  are  urea  nitrogen,  uric 
acid,  creatinine,  creatine,  sugar,  chlorides  in  the  form  of  sodium 
chloride,  and  cholesterol.  The  normal  amounts  of  these  con- 
stituents are  as  follows: 

Xonprotein  nitrogen  25  to  30  mgms.  per  100  c.c.  blood 

Urea  nitrogen  12  "    15        "        "      "      "       " 

Uric  acid  1  "      3.0     "        "     "     "       " 

Creatinine  1  "     2.5     "        "      "      "        " 

Creatine  5  "    10        "        "      "      "        " 

Sugar  0.08-0.12% 

Chlorides   as   sodium  chloride                    0.65% 

Cholesterol  >     0.15% 

For  purposes  of  comparison  we  refer  to  the  table  showing  val- 
ues in  various  diseases  (Fig.  63,  page  202)  in  which  we  tabulate 
the  normal  findings  and  the  changes  met  with  in  the  common  dis- 
eases. We  would  also  refer  the  reader  to  Fig.  64,  page  203,  show- 
ing nonprotein  nitrogen,  etc.,  in  which  more  elaborate  figures  are 
shown. 

At  this  point  we  wish  to  refer  to  the  significance  of  nonnitrog- 
enous  metabolism : 

Total  Nitrogen  is  eliminated  in  the  proportion  of  15  grams  per 
diem.  It  leaves  the  body  as  follows : 

Urea   (grams)  25  (12  gm.  N)     or  85% 

Creatinine             1.5  or  5% 

Uric  acid              0.5  or  2% 

Ammonia  s            0.5  or  4% 

Best  nitrogen      0.5  or  5% 


202 


BLOOD   AND   URINE    CHEMISTRY 


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BLOOD    CHEMISTRY   AND    NEPHRITIS 


203 


Fig.  64. 


204  BLOOD   AND   URINE    CHEMISTRY 

Where  does  urea  come  from?  In  digestion  protein  matter  is 
broken  down  into  amino-acids  which  are  picked  up  by  the  blood 
just  as  pieces  of  metal  are  picked  up  by  a  magnet.  Some  of  the 
amino-acids  are  retained  and  others  are  transformed  into  am- 
monia and  eliminated.  The  greater  part  of  the  nitrogen  that  is 
eliminated  is  exogenous  (coming  from  food)  and  its  elimination 
occurs  in  the  form  of  urea.  The  blood  holds  up  the  carbonates 
and  preserves  its  neutrality  by  this  means,  by  eliminating  or  get- 
ting rid  of  the  acids.  The  greater  part  of  the  acids  in  urine  are 
made  up  of  acid  phosphates,  derived  from  the  blood.  When  the 
blood  is  no  longer  able  to  get  rid  of  its  acids,  it  calls  upon  its 
ammonia  for  help.  This  has  already  been  alluded  to  in  the  chap- 
ter on  acidosis  (see  page  170).  The  determining  factor  in  re- 
spect to  nitrogen  in  urine  is  the  neutrality  of  the  blood.  If  you 
administer  enough  alkali,  you  can  cause  the  nitrogen  to  entirely 
disappear.  It  is  a  well-known  fact  that  rabbits  eliminate  no  nitro- 
gen in  their  urine  because  they  live  on  a  diet  that  contains  a  good 
deal  of  carbonates.  Nitrogen  depends  upon  the  hydrogen-ion  con- 
centration of  bodily  tissues. 

The  source  of  creatinine  is  entirely  endogenous.  It  is  con- 
stant day  by  day  in  the  body. 

There  have  been  some  interesting  data  experimentally  obtained 
as  to  the  effect  of  the  administration  of  creatine  and  creatinine 
to  animals.  Folin1  was  the  first  by  means  of  his  colorimetric 
methods  to  show  that  the  quantitative  conversion  of  creatine 
or  creatinine  to  creatine  in  vitro  was  far  more  difficult  than  pre- 
vious statements  would  lead  one  to  believe.  He  was  unable  to 
prove  that  feeding  experiments  with  creatine  in  man  were  fol- 
lowed by  conversion  into  creatinine.  Other  experimental  observa- 
tions were  made  by  Klercker,2  Wolf  and  Shaffer,3  van  Hoogen- 
huyze  and  Verploegh;4  and  others.  Myers  and  Fine5  conclude 
from  their  experimental  observations  that  the  administration  of 
creatinine  appears  to  exert  a  slight  increase  on  the  muscle  con- 
tent of  creatine.  When  creatinine  was  administered  an  average 
of  80  per  cent  appeared  in  the  urine  but  no  elimination  of  crea- 


rPolin:     Hammarsten's  Festschrift,   1906,  vol.   iii. 

ol    iii         45    Behr'    Z'    PhyS'    "'    Path"    19°6'   V°L    vii''    P-    59;    Bi-- 

"Wolf  and  Shaffer:     Jour.   Biol.   Chem.,   1908,  vol.   iv,  p.  489 
van  Hoogenhuyze  and  Veroloegh:     Ztschr.  f.  phys.  Chem.,   1908,  vol.  Ivii, 
°Myers  and  tme:     Jour.  Biol.  Chem.,  1913,  vol.  xvi,  p.  169. 


iochem-    Ztschr.,    1907, 
p.   161. 


BLOOD    CHEMISTRY   AND   NEPHRITIS  205 

tine  was  detected.  Folin  and  Denis6  experimentally  failed  to 
show  any  creatinine  formation  from  the  administration  of  crea- 
tine, although  they  noted  a  slight  accumulation  of  creatinine  in 
the  blood  and  a  slight  diminution  in  the  muscle.  In  a  later  paper 
Myers  and  Fine7  reiterate  their  belief  in  the  uniformity  obtained 
from  the  creatine  content  of  the  muscle  of  certain  animals,  par- 
ticularly the  rabbit,  and  suggest  that  this  might  ultimately  be 
found  to  be  the  underlying  factor  in  the  constancy  in  the  excre- 
tion of  creatinine.  Their  results  have  been  confirmed  by  Dor- 
ner,8  Mellanby,9  Riesser,10  Palladin  and.Wallenburger,11  and  Bau- 
mann.12 

It  appears  to  be  fairly  well  established,  therefore,  that  creatin- 
ine resides  in  muscle  and  that  it  is  constantly  present  in  blood 
in  about  the  same  quantity  at  all  times  in  health  in  the  adult. 
The  importance  of  creatinine  in  routine  blood  chemical  analysis 
in  connection  with  chronic  nephritis  has  also  been  very  well  estab- 
lished. It  seems  strange  that  for  so  long  a  time  only  estimations 
of  total  nonprotein  nitrogenous  blood  constituents  were  the  or- 
der of  the  day.  At  the  present  time  there  is  no  one  ingredient 
that  is  more  important  to  estimate  than  is  creatinine.  Cases  of 
blood  retention  of  which  uremia  constitutes  the  most  striking 
type,  show  accumulation  of  creatinine  as  well  as  urea  nitrogen  and 
uric  acid. 

Shaffer  has  shown  that  it  is  constant  hour  by  hour.  It  is  not 
materially  increased  by  protein  food  intake.  It  is  always  present 
in  muscle  tissue,  as  shown  by  Shaffer,13  and  Myers  and  Fine.14 
Myers  and  Fine15  believe  that  the  urinary  creatinine  is  originated 
from  muscle  tissue.  These  authorities16  have  published  a  num- 
ber of  observations  on  the  metabolism  of  creatine  and  creatinine. 
Their  paper  on  "The  Presence  of  Creatinine  in  Muscle"  shows 
the  content  of  creatinine  in  fresh  muscle  in  quantities  varying 


6Folin  and  Denis:     Tour.  Biol.  Chem.,   1914,  vol.  xvii,  p.  493. 

TMyers  and   Fine:     Jour.   Biol.   Chem.,   1915,  vol.  xxi,  p.   289. 

sDorner:     Ztschr.   f.   phys.    Chem.,   1907,  vol.   Hi,  p.   259. 

°Mellanby:     Jour.   Physiol.,   1907-8,  vol.  xxxvi,  p.   447. 

10Riesser:     Ztschr.  f.  phys.  Chem.,  voL  Ixxxvi,  p.  444. 

"Palladin  and  Wallenburger:     Compt.  rend.  Soc.  de  biol.,  1915,  vol.  Ixxviii,  p.  111. 

12Baumann:     Jour.  Biol.  Chem.,  1914,  vol.  xvii,  p.   15. 

"Shaffer:     Am.  Jour.   Physiol.,   1908-9,  vol.  xxiii,  p.   4. 

"Myers  and  Fine:     Am.  Jour.  Med.  Sc.,  1910,  vol.  cxxxix,  p.  256. 

"Myers  and  Fine:     Jour.   Biol.   Chem.,   1913,  vol.   xiv,  p.   24. 

16Myers  and  Fine:  Jour.  Biol.  Chem.,  1913,  vol.  xv,  p.  304;  Ibid.,  1913-14,  vol.  xvi, 
p.  174;  Ibid.,  1914,  vol.  xvii,  p.  65;  Proc.  Soc.  Exper.  Biol.  and  Med.,  1913,  vol.  xi,  p.  15; 
Ibid.,  1915,  vol.  xxi.  No.  2,  p.  383. 


206  BLOOD   AND   URINE    CHEMISTRY 

in  the  cases  of  rabbits  from  4.5  to  5.9  mgms.,  5.7  in  human  muscle 
in  leg  amputated  for  sarcoma,  2.6  in  leg  amputated  for  gangrene, 
6.8  in  pectoral  muscle  of  case  of  interstitial  nephritis,  6.8  in  heart 
muscle  from  uremic  case,  18.1  in  psoas  muscle  in  interstitial  neph- 
ritis. They  showed,17  as  did  Shaffer,18  and  Folin  and  Denis,19  that 
the  quantity  of  creatinine  present  in  the  muscle  is  much  greater 
than  that  of  the  blood,  liver  or  any  other  tissue.  The  fact  that 
the  greater  portion  of  the  preformed  creatinine  present  in  the 
body  is  found  in  the  muscle,  strongly  suggests  that  this  is  the 
chief  creatinine-forming  tissue. 

Uric  acid  is  partly  exogenous  and  partly  endogenous;  partly 
from  the  metabolism  of  food  and  partly  from  that  of  our  own  tis- 
sues. This  is  about  half  and  half.  If  liver  is  eaten,  we  can  raise 

HN-C-O 
NHC       i-NH 


HN-C=0  HN-C=0  HN-00 

NH 


71  /I 

O=C     C- 


^NH?-0 


Hypoxa-ntKirx  XantKin, 


the  amount  of  uric  acid  present.  It  comes  from  purin,  then 
changed  to  xanthin,  and  then  to  uric  acid.  It  has  to  be  de-amino- 
ized  before  change  to  xanthin  takes  place.  This  takes  place  by 
hypoxanthin  being  formed  from  adenin,  and  xanthin  is  formed 
from  guanin. 

The  following  graphic  representation  shows  this : 
The  second  part  of  the  process  is  an  oxidation,  i.  e.,  the  con- 
version 'of  hypoxanthin  into  xanthin  and  this  conversion  into  uric 
acid.     Uric  acid,  therefore,  is  the  chief  end-product  in  man  of 
nucleo-protein  metabolism. 

Uric  acid  is  a  difficult  substance  t^  dissolve.    It  is  soluble  1  part 

"Myers  and  Fine:     Jour.   Biol.   Chem.,   1915,  vol.  xxi,  p.   389. 
"Shaffer:     Jour.   Biol.  Chem.,   1910,  vol.  vii,  pp.   23,  30 
19Folm  and  Dems:     Jour.  Biol.   Chem.,  1914,  vol.  xvii,  p.  501. 


BLOOD    CHEMISTRY   AND    NEPHRITIS  207 

in  39  of  pure  water.  Urates  are  soluble  in  1  part  in  500  under 
conditions  as  they  exist  in  the  body.  Uric  acid  is  the  most  difficult 
for  the  kidney  to  excrete  of  the  nonprotein  blood  constituents ; 
urea  comes  next,  and  creatinine  last.  Expressed  in  other  terms, 
creatinine  is  the  easiest  constituent  for  the  kidneys  to  eliminate, 
urea  is  the  next,  and  uric  acid  is  the  last  to  be  eliminated.  Again, 
urea  exists  in  the  body  in  twenty  times  as  much  concentration  as 
creatinine  and  it  therefore  takes  twenty  times  as  much  work  for 
the  kidney  to  eliminate  its  urea  as  its  creatinine. 

With  these  fundamental  facts  before  us,  let  us  consider  what 
has  been  done  in  the  past  with  respect  to  studying  from  a  diag- 
nostic standpoint  the  character  of  nonprotein  metabolism  in  dis- 
ease. It  might  be  mentioned  in  passing  that  the  estimation  of  the 
kidney  function  has  long  been  considered  a  favorite  method  of  de- 
termination of  metabolic  faults.  For  instance,  the  indigo-car- 
min  test,  the  phlorizin  test  and  cryoscopy  of  blood  and  urine 
each  have  had  their  vogue  and  have  been  practically  abandoned 
because  of  the  meager  information  obtainable  thereby.  Possibly 
Geraghty  and  Rowntree,20  with  their  phenolsulphonphthalein  test, 
did  more  to  advance  the  cause  of  kidney  functional  tests  than 
any  of  their  predecessors.  This  test  of  kidney  function  is  quite 
reliable  but  it  has  its  limitations.  It  is  an  excellent  method  of 
estimating  the  function  of  the  kidney  for  the  moment  but  does 
not  represent  the  condition  of  the  kidneys  so  far  as  retention  of 
objectionable  constituents  are  concerned,  over  a  long  enough 
period  of  time  to  accurately  weigh  bodily  metabolic  changes  in 
nonprotein  nitrogen. 

The  question  of  the  comparative  value  of  the  Geraghty  and 
RowTntree  test  and  the  blood  chemical  analysis  for  nonprotein 
nitrogenous  constituents  \vas  experimentally  carried  out  by  Froth- 
ingham,  Fitz,  Folin,  and  Denis.21  Rabbits  were  used  and  experi- 
mental nephritis  produced  by  the  injection  of  uranium  nitrate 
(1.25  to  3  mgms.)  subcutaneously.  The  first  series  of  animals 
were  killed  under  anesthesia  by  bleeding  from  the  carotid  ar- 
teries. They  were  killed  on  consecutive  days  from  one  to  ten 
days  after  administration  of  uranium  nitrate.  In  a  second  series 


20Geraghty  and  Rowntree:     Jour.  Pharm.  and  Exper.  Therap.,  1910,  vol.  i,  p.  579. 
21Frothingham,  Fitz,  Folin  and  Denis:     Arch.  Int.  Med.,  1913,  vol.  xii,  p.   145. 


208  BLOOD   AND   URINE    CHEMISTRY 

of  experiments  the  animals  were  allowed  to  recover,  and  the 
blood  chemical  analyses  and  the  phenolsulphonphthalein  tests  were 
made  periodically.  The  blood  specimens  for  chemical  analyses 
were  taken  from  the  marginal  ear  veins.  The  rabbits  were  kept 
in  cages,  fed  on  carrots  and  hay,  100  grams  of  carrots  per  day, 
with  50  c.c.  of  water  administered  by  means  of  a  stomach  tube 
before  the  injection  of  the  phenolsulphonphthalein  (1  c.c.  con- 
taining 6  mgms.)  into  the  muscles  of  the  thigh.  The  animals 
were  kept  in  a  small  cage  over  a  glass  funnel  to  prevent  loss  of 
urine.  After  70  minutes  the  urine  was  obtained  by  massage. 
The  determination  was  made  according  to  Geraghty  and  Rown- 
tree's  method  (see  page  89).  It  was  seen  that  the  normal  rabbits 
have  about  30  mgms.  of  urea  nitrogen  per  100  c.c.  The  rate  of 
phenolsulphonphthalein  in  excretion  in  normal  rabbits  is  about 
60  per  cent  in  70  minutes. 

These  experimental  observations  on  uranium  nephritic  rabbits 
showed  a  decrease  in  the  excretion  of  phenolsulphonpththalein 
and  a  great  accumulation  of  nonprotein  nitrogenous  constituents. 
The  decrease  in  the  phenolsulphonphthalein  amounted  to  as  little 
as  a  trace  only.  The  retention  of  nonprotein  nitrogen  amounted  to 
as  much  as  216  mgms.  and  of  ureas  as  much  as  172  mgms.  The 
retention  of  nitrogen  remained  high  even  where  the  phenolsul- 
phonphthalein began  to  improve.  In  general  the  tests  paralleled 
each  other.  In  another  series  of  experiments,  the  blood  was  col- 
lected every  two  or  three  days  from  the  veins.  The  nitrogen 
seemed  to  go  on  being  retained  even  where  the  phenolsulphon- 
phthalein excretion  was  improving.  This  seemed  to  prove  that 
the  nitrogenous  retention  represented  the  difference  between  that 
eliminated  and  that  produced,  whereas  the  phenolsulphonphtha- 
lein is  an  indicator  of  elimination  alone.  This  represents  essen- 
tially the  difference  between  the  two  tests.  The  percentage  of 
phenolsulphonphthalein  excreted  affords  an  index  of  the  kidney 
function  at  the  time  the  test  is  made.  The  result  is  apparently 
not  at  all  influenced  by  the  length  of  time  the  kidney  may  have 
been  in  the  condition  indicated  by  the  test.  In  general  these 
tests  parallel  each  other  as  indicators  of  kidney  function  with  t-he 
essential  difference,  however,  that  the  amount  of  phenolsulphon- 
phThalein  excretion  shows  the  renal  function  for  the  moment.  The 


BLOOD    CHEMISTRY   AND   NEPHRITIS  209 

amount  of  nonprotein  nitrogen  and  urea  nitrogen  in  the  blood 
is  rather  a  measure  of  accumulating  difference  between  the  waste 
nitrogen  produced  in  metabolism  and  amount  eliminated  by  the 
kidneys.  The  time  element,  the  duration  of  the  condition,  is 
therefore  an  important  factor  in  weighing  up  to  these  results. 
The  outcome  of  a  case  cannot  be  estimated  nearly  so  well  by 
functional  kidney  tests  as  by  blood  chemical  analyses.  Foster22 
reported  a  case  of  marked  kidney  disease  with  normal  elimination 
of  phenolsulphonphthalein.  If  the  prognosis  had  been  based  upon 
the  phenolsulphonphthalein  output,  this  patient  would  have  re- 
covered, but,  as  a  matter  of  fact,  he  died.  Again,  he  mentions  the 
fact  that  a  low  output  would  not  indicate  a  fatal  termination  in 
cases  of  chronic  nephritis.  In  Foster's  case  with  an  output  of 
28  the  patient  died  within  two  days  in  coma.  It  can  thus  be  seen 
that  a  normal  output  of  phenolsulphonphthalein  does  not  neces- 
sarily indicate  kidney  lack  of  function  insofar  as  nonprotein  ni- 
trogenous retention  is  concerned,  nor  does  a  lower  output  than 
normal  indicate  the  outcome  of  p,  case.  It  will  be  seen  later  that 
we  have  in  the  estimation  of  the  creatinine  values  particularly, 
a  very  valuable  means  of  prognosis. 

Assuming,  therefore,  that  the  moment  is  now  at  hand  in  diag- 
nosis, where  we  must  weigh  up  the  character  of  blood  retention  in 
cases  of  nephritis,  it  is  manifest  that  the  blood  chemical  figures 
are  the  most  trustworthy  that  can  be  gathered.  We  have  noted 
already  the  percentage  of  nonprotein  nitrogenous  concentrations 
in  health.  In  degenerative  conditions  of  the  kidney,  these  blood 
constituents  are  markedly  altered.  In  early  interstitial  nephritis, 
we  have  the  beginning  of  retention  in  the  shape  of  an  accumulation 
of  but  one  ingredient,  namely,  uric  acid.  Here  values  may  be  seen 
as  high  as  from  4  to  6  mgms.  per  10  c.c.  of  blood,  as  opposed  to 
the  normal  values  of  1  to  3.0  mgms.  Next  we  have  in  more  ad- 
vanced cases  an  accumulation  of  creatinine  in  the  blood,  the  figure 
2.5  mgms.  representing  the  upper  limit  of  the  normal  and  any 
figure  over  this  constituting  an  abnormality.  This  accumulation 
of  the  three  constituents  in  their  order,  uric  acid  first,  urea  sec- 
ond and  creatinine  third,  represents  the  fact  already  detailed, 
that  uric  acid  is  the  most  difficult  substance  for  the  kidney  to  ex- 

^Foster,  N.  B. :     Arch.  Int.  Med.,   1913,  vol.  xii,  p.  452. 


210  BLOOD   AND   URINE    CHEMISTRY 

crete;  urea  occupying  an  intermediate  position,  while  creatinine 
is  the  easiest.  We  have  alluded  before  to  the  "stair-case"  effect 
of  retention  first  pointed  out  by  Myers  and  Fine.  Chace  and 
Myers23  give  a  tabulated  list  of  cases  showing  this  effect  (see 
Table  XIII,  page  211. 

It  can  readily  be  seen  from  this  table  that  the  first  accumula- 
tion in  the  blood  when  kidney  function  is  interfered  with  by  be- 
ginning chronic  interstitial  nephritis  is  in  the  uric  acid  values, 
next  there  occurs  an  accumulation  of  urea  as  well  as  uric  acid, 
and  finally,  in  uremic  nephritis  we  have  an  accumulation  of  uric 
acid,  urea  nitrogen,  and  creatinine.  This  seems  particularly  in- 
teresting and  important  in  view  of  the  fact  that  the  urinary 
changes  in  some  of  these  cases  are  exceedingly  scant.  The  find- 
ing of  albumin  and  casts  is  often  made,  but  this  gives  the  clinician 
but  little  information  as  regards  the  true  metabolic  processes 
that  are  going  on  and  the  exact  state  of  kidney  function.  We  can- 
not well  understand  how  a  clinician  can  safely  pass  judgment 
in  a  case  of  chronic  nephritis  without  an  examination  of  the  blood 
for  these  ingredients. 

To  recapitulate,  we  know  that  the  greatest  amount  of  reten- 
tion of  urea,  uric  acid,  and  creatinine  occurs  in  chronic  inter- 
stitial nephritis  particularly  when  uremia  is  at  hand.  A  prog- 
nostic sign  of  no  mean  importance  is  that  first  pointed  out  by 
Myers  and  Lough21*  in  their  paper  on  "Diagnostic  Value  of 
Creatinine  in  the  Blood  in  Nephritis."  They  showed  at  that 
time  (1915)  that  when  creatinine  in  the  blood  appeared  in  the 
concentration  of  5  mgms.  per  100  c.c.  of  blood  and  over,  that 
every  one  of  these  cases  terminated  fatally.  Of  the  eleven  cases 
in  their  series  showing  over  5  mgms.  of  creatinine  per  100  c.c.  of 
blood,  all  terminated  fatally  in  from  a  few  days  to  two  months. 
In  this  group  of  cases  the  phenolsulphonphthalein  output  was 
practically  zero,  with  but  one  exception.  These  cases  of  creatinine 
values  of  5  mgms.  or  above  were:  a  case  of  mercuric  bichloride 
poisoning,  with  creatinine  value  of  33.3  mgms. ;  a  case  of  chronic 
interstitial  nephritis  in  uremia  with  creatinine  of  20.5  mgms. ;  six 
other  cases  of  interstitial  nephritis,  with  creatinine  values  of  20.0, 

23Chace  and  Myers:     Jour.  Am.  Med.  Assn.,  1916,  vol.  Ixvii,  No.  13,  p.  929. 
24Myers.and  Lough:     Arch.  Int.  Med.,  1915,  vol.  xvi,  pp.  536-546. 


BLOOD    CHEMISTRY    AND    NEPHRITIS 


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212  BLOOD   AND   URINE    CHEMISTRY 

16.7,  16.6,  14.7,  11.0  and  5.3  mgms.  respectively;  three  cases  of 
chronic  diffuse  nephritis,  with  uremia,  with  creatinine  values  of 
14.7,  7.4,  and  7.0  mgms.  of  creatinine  respectively.  They  have 
three  times  as  many  cases  on  record  in  which  this  fact  was  borne 
out. 

The  prognostic  value  of  the  finding  of  5  mgms.  of  creatinine 
or  over  has  been  confirmed  by  the  writers,  together  with  Schisler, 
in  a  group  of  cases  of  thermic  fever  recently  studied  at  the  St. 
Louis  City  Hospital,  a  full  report  of  which  will  be  shortly  pub- 
lished. Here  we  had  a  set  of  blood  findings  identical  in  all  par- 
ticulars with  those  of  uremia.  We  present  in  Fig.  65  a  tabulated 
picture  of  these  cases,  showing  their  blood  and  urinary  findings. 

We  are  able  to  record  three  cases  of  thermic  fever  in  which  the 
creatinine  values  of  4.8,  5.0,  and  6.1  mgms.  respectively,  pointed 
to  a  fatal  ending,  which  ensued  within  forty-eight  hours  from  the 
time  when  the  record  was  made.  In  the  case  of  0 'Conner,  the 
observation  was  made  on  August  1,  and  the  patient  died  the  same 
day.  He  showed  urea  nitrogen  of  33  mgms.,  uric  acid  13.2, 
creatinine  4.8  (slightly  below  the  fatal  prognostic  point),  and 
blood  sugar  0.15%.  His  urine  analysis  showed  albumin  and 
coarsely  granular  casts.  The  next  case  (Fischer)  ran  rather  a 
long  course  for  a  case  of  thermic  fever  which  was  from  the  out- 
set quite  severe.  This  individual  entered  the  hospital  on  August 
2,  1916,  showing  a  severe  picture,  semiconscious,  rise  in  temper- 
ature to  108°  F.,  delirium.  His  blood  findings  on  the  first  day 
were  urea  nitrogen  32,  uric  acid  8.6,  and  creatinine  4.1  mgms. 
From  day  to  day  he  was  tested  and  showed  at  first  a  slight  decline 
in  his  blood  findings.  On  the  eighth  day  of  his  stay  in  the  hos- 
pital his  creatinine  reached  the  fatal  point  of  5.0  mgms.  He  died 
two  days  later.  Autopsy  on  this  case  showed  simply  cloudy  swell- 
ing of  the  kidneys  and  no  other  gross  changes  anywhere.  It 
might  be  mentioned  that  his  Wassermann  of  blood  and  spinal 
fluid  was  negative.  His  urinary  findings  during  all  this  time 
showed  at  first  a  very  heavy  amount  of  albumin  and  moderate 
number  of  granular  casts.  Towards  the  end  of  life  the  urine 
cleared  up  as  regards  albumin,  but,  on  the  day  before  death,  the 
microscopical  picture  showed  the  fields  actually  crowded  with 
granular  casts.  The  next  two  cases  (Huth  and  Ship)  are  especi- 


BLOOD    CHEMISTRY    AND    NEPHRITIS 


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214  BLOOD   AND   URINE    CHEMISTRY 

ally  interesting  in  that  the  one  case  (Huth),  with  an  apparently 
hopeless  clinical  symptomatology,  had  a  very  good  blood  picture 
(urea  nitrogen  26,  uric  acid  9.6,  creatinine  3.83  mgms.)  while 
the  other  case  (Ship),  observed  at  the  same  time,  with  a  much 
more  favorable  clinical  picture,  showed  a  very  grave  set  of  blood 
findings;  viz.,  urea  nitrogen  76,  uric  acid  14.8,  and  creatinine  6.1 
mgms.  In  the  Huth  case  an  unfavorable  clinical  prognosis  was 
made,  but  a  good  prognosis  was  issued  after  the  blood  examina- 
tion was  completed.  True  to  the  latter  prediction,  he  promptly 
recovered.  The  second  case  with  a  rather  favorable  clinical  prog- 
nosis was  condemned  by  the  blood  findings  of  creatinine  over  5 
mgms.  True  to  this  prediction,  he  died  on  the  following  morning. 
Both  cases  showed  substantially  the  same  urinary  findings,  thus  il- 
lustrating that  no  prognostic  record  could  accurately  be  made  in 
this  way.  The  last  case  was  observed  and  tested  during  the 
period  of  his  convalescence  and  showed  almost  normal  findings. 
These  cases,  therefore,  served  to  illustrate  the  great  value  of 
blood  chemical  methods  in  first  demonstrating  that  the  condition 
met  with  in  thermic  fever  is  quite  analogous  to  that  seen  in 
uremic  nephritis,  secondly,  in  proving  Myers,  Lough  and  Chace's 
contention  that  the  finding  of  over  5.0  mgms.  of  creatinine  in 
blood  serves  to  indicate  a  fatal  ending  in  any  case.  A  report  of  a 
most  unusual  case  of  chronic  interstitial  nephritis,  with  findings 
in  blood  and  urine  made  by  Halsey,25  serves  as  an  object  lesson 
in  pointing  out  the  value  of  this  type  of  work.  This  patient  was 
well  enough  to  visit  the  observer's  office  with  symptoms  of  a  sub- 
jective nature  so  slight  as  to  be  almost  incompatible  with  the  find- 
ings on  physical  examination  and  blood  analyses  and  subsequent, 
rapid,  fatal  ending.  He  was  on  his  way  to  Florida,  but  stopped 
off  in  New  York  with  but  little  idea  evidently  of  the  seriousness 
of  his  condition.  His  urine  showed  no  albumin  or  casts.  His 
blood  examination  showed  urea  nitrogen  97,  uric  acid  6.6,  creatin- 
ine 17.5,  blood  sugar  0.18  per  cent,  blood  plasma  combining  power 
50.  Because  of  these  desperate  findings  he  was  further  detained 
and  carefully  observed.  After  three  days  of  nitrogen-poor  diet, 
the  blood  examination  showed  urea  nitrogen  129  mgms.,  uric  acid 
6.3,  creatinine  21.8,  blood  sugar  0.18  per  cent.  His  nitrogen  in- 

25Halsey,  R.  H.:    Jour.  Am.  Med.  Assn.,  June  10,  1916,  vol.  Ixvi,  No.  24,  p.   1847. 


BLOOD    CHEMISTRY   AND    NEPHRITIS  215 

take  was  restricted,  and  seven  days  later  his  findings  were:  urea 
nitrogen  132,  uric  acid  7,  and  creatinine  22.3,  with  an  increase  in 
the  carbon  dioxide  combining  power  of  his  blood  plasma  to  53. 
Four  days  later,  still  on  nitrogen-poor  diet,  he  showed  urea  ni- 
trogen 144,  uric  acid  6.1,  and  creatinine  28.9.  His  carbon  dioxide 
combining  power  was  diminished  to  50.  His  protein  diet  was  here 
increased  owing  to  the  effect  on  the  tissues  of  too  long  an  ab- 
stinence from  nitrogenous  food.  Three  days  later  the  findings 
were  urea  nitrogen  150,  uric  acid  5.6,  creatinine  24.2,  blood  sugar 
0.20  per  cent  and  carbon  dioxide  down  to  33.  Further  blood  ex- 
aminations showed  a  corresponding  rise  in  blood  constituents 
and  death  of  patient  occurred  on  the  twenty-fifth  day  of  his  ob- 
servation. This  patient  showed  physically  a  picture  of  hyper- 
tension with  but  the  slightest  hypertrophy  of  the  heart.  The 
conclusions  of  Halsey  from  this  record  were  very  aptly  stated; 
i.  e.,  that  with  the  examination  of  the  urine  only,  the  seriousness 
of  the  patient's  condition  would  not  have  been  discovered,  also 
that  while  the  phenolsulphonphthalein  test  was  of  value  in  indi- 
cating the  status  of  the  patient  for  the  moment,  the  amount  of 
urea  and  creatinine,  particularly  the  latter,  gave  the  best  clue 
as  to  the  progress  and  the  prognosis. 

Another  set  of  conditions  in  which  the  blood  chemical  analysis 
should  prove  of  striking  value  to  the  clinician  is  the  group  of 
cases  called  cardio-vascular  with  only  secondary  renal  disturbance. 
Differentiation  of  these  cases  from  cases  of  true  nephritis  with 
secondary  cardiac  and  blood  vessel  change  might  well  be  made 
by  means  of  the  colorimetric  methods.  Through  the  courtesy  of 
Dr.  Edwin  Schisler  of  the  St.  Louis  City  Hospital  Staff,  we  are 
permitted  to  record  some  data  on  this  group  of  cases  (see  Table 
XIV). 

It  will  be  readily  seen  that  in  these  cases  which  showed  the  symp- 
tomatology of  mixed  cardiac  and  renal  disease,  there  was  little 
if  any  retention  of  the  nonprotein  nitrogenous  ingredients  in 
blood.  The  importance  of  blood  chemical  analyses  in  this  vari- 
ety of  clinical  condition  can  well  be  appreciated. 

Test  Meal  for  Renal  Function  and  Ambard  Coefficient. 
Besides  the  well  known  phenolsulphonphthalein  functional  kid- 


216 


BLOOD   AND   URINE    CHEMISTRY 
TABLE  XIV 


UREA  NITROGEN 

TJRIC  ACID 

CREATININE 

NAME 

Mgms.  per  100 
c.c.  of  Blood 

Mgms.perlOO 
c.c.  of  Blood 

Mgms.perlOO 
c.c.  of  Blood 

"D" 

7/28/16 

c? 

13 

3.2 

2.7 

"B" 

8/1/16 

cf 

12 

2.8 

2.8 

"S" 

8/2/16 

cf 

12 

1.0 

2.8 

"M" 

8/10/16 

c? 

12                        2.4 

2.7 

*  <?  Male. 
6   Female. 

ney  test  and  the  estimation  of  urea  nitrogen,  uric  acid,  creatinine, 
and  sugar  in  blood,  there  are  other  measures  of  estimation  of 
bodily  metabolism  as  respect  kidney  function.  A  work  of  this 
kind  would  be  incomplete  if  these  were  omitted.  The  other  two 
methods  which  are  used  for  certain  definite  reasons  are  those 
known  as  the  Ambard  coefficient  of  urea  excretion,  and  the  test 
meal  for  renal  function. 

The  renal  test  meal  and  the  estimation  of  renal  function  by 
this  means  is  exceedingly  simple  in  hospital  practice  but  difficult 
to  carry  out  in  private  practice.  The  urine  is  collected  every 
two  hours  during  the  day,  while  the  patient  is  on  a  full  diet,  and 
a  ten  to  twelve  hour  specimen  is  collected  at  night.  No  food  or 
drink  is  taken  except  at  meal  times.  The  collection  of  the  night 
specimen  is  begun  three  hours  after  the  evening  meal.  A  normal 
test  yields  a  maximum  specific  gravity  of  1018  or  more.  The 
specific  gravity  varies  but  nine  points  or  more  from  the  highest 
to  the  lowest  figure,  and  the  night  urine  is  small  in  amount,  400 
c.c.  or  less  and  of  high  specific  gravity,  1018  or  over.  A  lowering 
of  the  maximum  specific  gravity,  a  fixation  of  the  specific  gravity 
and  a  nocturnal  polyuria  are  the  signs  indicative  of  diminished 
renal  function. 

Mosenthal  and  Lewis26  have  given  us  an  excellent  account  of 
these  two  measures  as  compared  to  the  Geraghty  and  Rowntree 
test  and  the  estimation  of  the  nonprotein  nitrogenous  constituents 
in  blood.  They  insist  upon  regarding  each  one  of  these  measures 
as  particularly  designed  to  cover  certain  characteristics  of  each 

26Mosenthal  and  Lewis:  Jour.  Am.  Med.  Assn.,  Sept.  23,  1916,  vol.  Ixvii,  No.  113, 
p.  933. 


BLOOD    CHEMISTRY'  AND   NEPHRITIS  217 

case  and  speak  of  them  seriatim.  Each  has  its  place,  each  its  in- 
dication, and  from  each  valuable  deductions  may  be  drawn.  The 
Ambard  coefficient  of  urea  excretion  expresses  numerically  the 
relation  between  the  concentration  of  urea  in  blood  and  the  rate 
of  excretion  of  urea  in  the  urine.  As  a  result  of  the  study  of 
normal  human  beings,  Ambard27  has  asserted  that  when  the  con- 
centration of  urea  in  the  urine  is  constant,  the  quantity  of  urea 
excreted  in  the  urine  varies  proportionately  to  the  square  root 
of  the  concentration  of  urea  in  the  blood  ;  thus  : 

Urea  in  Mood        =  Constant?  or       Urea  in  blood  _  ^^ 


A/ Excretion  per  unit  of  time 

Also,  when  the  concentration  of  urea  in  the  blood  remains  con- 
stant, the  quantity  excreted  in  the  urine  varies  inversely  as  the 
square  root  of  the  concentration  in  the  urine;  thus: 

Bate  of  excretion  I          -^/Concentration  II 

Eate  of  excretion  II  ~"     / — 

-\/ Concentration  I 

Or,  as  expressed  by  Mosenthal  and  Lewis: 

Ur 


\         P       25 

In  which  K  =  the  coefficient  of  urea  excretion. 
Ur  — urea  grams  per  liter  of  blood. 
D  =  urea  grams  excreted  in  urine  in  24  hours. 
C  — urea  grams  per  liter  of  urine. 
P  =  body  weight  in  kilograms. 
70  —  standard  body  weight  in  kilograms. 
25  =  standard  concentration  of  urea  grams  per  liter  of  urine. 

McLean  and  Selling28  have  controlled  Ambard 's  original  method 
by  using  the  exact  methods  of  Folin,  and  state  that  "  Ambard 's 
coefficient,  when  computed  from  results  obtained  by  the  accurate 
methods  of  Folin  and  his  collaborators,  varies  in  normal  persons 
only  between  comparatively  narrow  limits,  and  may  .be  regarded 
as  constant,"  and  further  "that  ingestion  of  urea  does  not  ma- 
terially alter  the  value  of  Ambard 's  coefficient,  provided  sufficient 


2IAmbard:     Physiologic  normale  et  pathologique  des  reins,  Paris,  1914. 
^McLean  and  Selling:     Jour.  Biol.  Chem.,  1914,  vol.  xix,  p.  31. 


218  BLOOD   AND   URINE    CHEMISTRY 

time  is  allowed  for  absorption  before  examination  is  made.  The 
normal  coefficient  is  between  0.06  and  0.09,  0.08  being  considered 
the  figure."  Quoting  from  Mosenthal  and  Lewis:29  "When  the 
values  rise  above  0.09,  some  impairment  of  the  power  of  the  kid- 
ney to  excrete  urea  is  indicated.  Inability  of  the  kidney  to  elimi- 
nate urea  in  proportion  to  the  concentration  of  the  blood  urea 
results  in  an  increase  in  proportion  to  the  concentration  of  the 
blood  urea  results  in  an  increase  in  Ambard's  coefficient.  In  a 
normal  individual  it  will  remain  within  the  limits  mentioned,  no 
matter  what  the  height  of  blood  urea;  in  cases  with  impaired 
renal  function,  however,  the  kidney  does  not  answer  the  diuretic 
stimulus  of  the  blood  urea  adequately,  too  little  urea  is  put  out, 
and  the  result  is  a  rising  coefficient,  whether  the  urea  in  the  blood 
be  high  or  low.  The  degree  of  the  impairment  of  renal  func- 
tion, as  indicated  by  the  various  levels  of  Ambard's  coefficient,  is 
indicated  in  Table  XV. 

"The  test  meal  for  renal  function  which  Mosenthal  and  Lewis 
refer  to  consists  in  the  two  hour  collections  of  urinary  speci- 
mens during  the  day,  while  the  patient  is  on  a  full  diet,  and  of 
a  ten  to  twelve  hour  specimen  at  night.  The  patient  is  given  no 
food  or  fluid  except  at  meal  times.  The  collection  of  the  night 
specimen  is  begun  three  hours  after  the  evening  meal.  Under 
these  circumstances,  a  normal  test  yields  a  maximum  specific 
gravity  of  1018  or  more,  the  specific  gravity  varies  9  points  or 
more  from  the  highest  to  the  lowest,  and  the  night  urine  is  small 
in  amount  (400  c.c.  or  less)  and  of  a  high  specific  gravity  (1018 
or  more).  These  criteria  are  the  same  as  those  originally  de- 
manded of  a  normal  test,  with  the  exception  that  a  difference  of 
9  degrees  between  the  highest  and  the  lowest  observations  has 
been  called  normal,  instead  of  10.  A  lowering  of  the  maximal 
specific  gravity,  a  fixation  of  the  specific  gravity  and  a  nocturnal 
polyuria  are  the  signs  indicating  a  diminished  renal  function. 

"Table  XV  gives  the  various  degrees  of  impairment  as  indi- 
cated by  the  test  meal  for  renal  function,  as  compared  with  the 
other  tests.  The  salt,  nitrogen,  and  other  urinary  constituents 
may  be  determined  in  these  specimens,  and  valuable  information 
may  be  obtained  as  to  the  ability  of  the  body  to  excrete  these 

^Mosenthal  and  Lewis:  Jour.  Am.  Med.  Assn.,  Sept.  23,  1916,  vol.  Ixvii,  No.  113, 
p.  933. 


BLOOD    CHEMISTRY    AND    NEPHRITIS 


219 


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220  BLOOD   AND   URINE    CHEMISTRY 

substances.  However,  the  simple  procedure  of  measuring  the 
volume  of  the  urine  and  determining  the  specific  gravity  yields 
sufficient  data  to  give  an  adequate  idea  of  renal  function  in 
many  respects,  and  the  quantitative  chemical  determinations  may 
be  resorted  to  when  more  detailed  information  is  desired.  In 
order  to  study  the  relation  to  one  another  of  the  evidences  of  im- 
paired renal  function  obtained  by  these  various  tests,  a  some- 
what arbitrary  scale  of  four  degrees  of  impairment ;  slight,  moder- 
ate, marked,  and  maximal,  was  determined  on.  The  exact  figures 
which  the  majority  of  experienced  observers  consider  as  indicat- 
ing normal  function,  and  these  various  degrees  of  subnormal 
function,  were  selected  and  the  findings  in  over  200  patients  were 
grouped  in  accordance  with  this  scale." 

The  contention  of  Mosenthal  and  Lewis  is  that  each  one  of 
these  methods  calls  attention  to  a  relative  degree  of  involvement 
of  kidney  function  and  that  each  one  of  them  has  a  significance 
apart  from  the  others.  They  conclude,  therefore,  that  a  compari- 
son according  to  this  method  is  an  extremely  valuable  aid  in  the 
treatment  and  prognosis  of  diseases  of  the  kidney.  They  cor- 
rectly assert  that  so  far  as  nonprotein  nitrogenous  retention  is 
concerned,  differentiation  must  be  made  in  weighing  the  results 
in  the  balance  between  kidney  efficiency,  diet  and  protein  destruc- 
tion. It  must  be  remembered,  however,  that  the  chemical  analysis 
of  blood  offers  perhaps  the  readiest  method  and  the  most  signifi- 
cant in  its  findings  over  all  other  methods  alluded  to  above.  We 
are,  therefore,  inclined  to  believe  that  the  renal  test  meal,  al- 
though of  exceedingly  great  utility,  cannot  approach  in  definite- 
ness  the  blood  chemical  tests.  So  far  as  the  estimation  of  Am- 
bard's  coefficient  is  concerned,  we  are  inclined  to  agree  with 
Chace  and  Myers30  that  this  method  gives  no  additional  informa- 
tion over  the  estimation  of  uric  acid,  urea  and  creatinine  of  the 
blood,  and  the  phenolsulphonphthalein  of  the  urine.  This  is  in 
line  with  the  conclusions  of  Addis  and  "Watanabe,31  that  the  rate 
of  urea  excretion  in  man  varies  under  physiological  conditions 
in  a  manner  which  cannot  be  explained  by  the  concentrations  of 
urea  in  the  blood  and  urine. 

The  value  of  the  Ambard  quotient  in  the  estimation  of  renal 

s»Chace  and  Myers:     Jour.  Am.  Med.  Assn.,  1916,  vol.  Ixvii,  No.   13,  p.  929. 
"Addis  and  Watanabe:     Jour.  Biol.  Chem.,  1916,  vol.  xxiv,  p.  203. 


BLOOD    CHEMISTRY   AND    NEPHRITIS  221 

function  has  more  recently  been  taken  up  by  Jonas  and  Austin.32 
They  call  attention  to  the  fact  that  in  addition  to  the  observations 
of  Addis  and  Watanabe,33  Pepper  and  Austin,3"*  in  dogs,  using, 
however,  total  nitrogen  instead  of  urea,  found  enormous  varia- 
tions in  the  quotient  in  different  animals  and  in  the  same  animal 
under  different  conditions.  These  two  investigators  studied  the 
Ambard  coefficient  as  modified  by  McLean  on  a  number  of  indi- 
viduals with  presumably  normal  kidneys  and  showed  that  the 
quotient  is  anything  but  constant.  In  this  study  which  was  made 
on  patients  in  the  medical  wrard  of  the  University  of  Pennsylvania 
Hospital,  periods  of  72  minutes  were  employed  (or  in  a  few  in- 
stances slightly  larger  periods  up  to  160  minutes),  and  the  blood 
withdrawn  from  the  arm  36  minutes  after  the  period  began.  The 
urea  was  determined  by  the  urease  method  of  Van  Slyke  and 
Cullen.35  Their  cases  were  divided  into  three  groups :  first, 
cases  in  which  there  was  no  clinical  or  laboratory  evidence  of 
nephritis,  nor  of  marked  cardiovascular  disease,  nor  of  cardiac 
decompensation ;  second,  cases  with  definite  evidence  of  more  or  less 
severe  nephritis;  third,  a  few  cases  in  which  there  was  no  definite 
nephritis,  but  in  which  there  was  more  or  less  vascular  disease 
or  cardiac  decompensation  or  both.  In  the  first  group,  there  was 
a  wide  variation  of  the  index  in  the  same  individual  on  different 
occasions  and  in  different  individuals.  The  conclusions  of  these 
observers  on  both  normal  and  abnormal  cases  were: 

1.  The  Ambard  formula  in  its  original   form  or  as  modified 
by  McLean  does  not  express  precisely  the  law  of  renal  function 
with  respect  to  the  elimination  of  urea,  and  this  is  particularly 
true  as  regards  the  effect  of  urinary  urea  concentration. 

2.  The  upper  limit  of  blood  urea  in  nonnephritic  and  normal 
individuals  under  ordinary  conditions  of  diet  and  life  is  about 
0.35  gm.  urea  per  liter  of  blood.     Figures  higher  than  this  are, 
under  ordinary  conditions  of  diet,  to  be  considered  evidence  of 
impaired  renal  function. 

3.  Using  McLean's  modification  of  Ambard 's  formula,  it  was 
found  that  in  the  great  majority  of  nephritic  cases  a  lowering  of 
the  index  was  accompanied  by  an  elevation   of  the  blood  urea 


32Jonas  and  Austin:     Am.  Jour.  Med.  Sc.,  October,  1916,  vol.  clii.  No.  4,  p.  560. 
83 Addis  and  Watanabe:     Jour.  Biol.  Chem.,  1916,  vol.  xxiv,  p.  203. 
^Pepper  and  Austin:     Jour.  Biol.   Chem.,   1915,  vol.  xxii,  p.  81. 
KVan  Slyke  and  Cullen:     Jour.  Biol.  Chem.,  1914,  vol.  xix,  p.  211. 


222  BLOOD   AND   URINE    CHEMISTRY 

above  normal  limits,  0.35  gm.  per  liter,  and  that  the  index  af- 
forded no  information  of  diagnostic  or  prognostic  value  that 
could  not  be  as  readily  deduced  from  the  blood  urea  alone. 

4.  In  certain  cases,  the  index  was  found  to  be  lowered  when 
the  blood  urea  was  within  normal  limits.     This  was  especially 
true  in  arteriosclerotic  cases  and  in  cases  with  cardiac  decompen- 
sation, which  probably  detracts  from  the  clinical  value  of  the  in- 
dex as  compared  with  that  of  the  blood  urea  rather  than  the  re- 
verse, since  it  is  of  importance  to  distinguish  between  cases  of 
vascular  and  renal  character. 

5.  In  the  determination  of  the  index  there  is  a  possibility  of 
error  arising  from  undetected  incomplete  collection  of  the  urine, 
which  cannot  occur  in  the  simple  blood  urea  estimation. 

6.  The  urea  index  estimated  repeatedly  in  the  same  individual 
exhibits  wider  variations  in  the  normal    or    nonnephritic    indi- 
vidual than  in  the  nephritic. 

7.  For  purposes  of  ordinary  clinical  diagnosis  and  prognosis 
the  estimation  of  blood  urea  is  a  more  reliable  and  more  useful 
guide  than  is  the  urea  index  or  the  Ambard  quotient. 

Blood  Sugar  and  Nephritis. 

Attention  must  be  called  to  the  fact  that  diabetes  may  often 
be  complicated  by  nephritis  and  that,  therefore,  the  study  of  blood 
chemistry  of  these  individuals  is  most  imperative.  The  presence 
of  undue  sugar  in  the  blood  and  urine  of  these  cases  calls  at- 
tention to  the  estimation  of  all  the  other  blood  ingredients  com- 
monly searched  for  in  nephritis.  It  must  also  be  remembered 
that  hyperglycemia  exists  in  severe  nephritis ;  this  has  been  recog- 
nized for  some  time  by  Bang,36  Neubauer,37  Roily  and  Opper- 
mann,38  and  Hopkins.39  Myers  and  Bailey40  allude  to  it  in  con- 
nection with  an  observation  of  a  number  of  hospital  cases.  So  we 
may  have  hyperglycemia  with  nephritis  and  nephritis  complicat- 
ing diabetes.  Severe  nephritis  seems  to  reduce  the  permeability 
of  the  kidney  for  sugar.  In  one  of  their  fatal  cases,  Myers  and 
Bailey  point  to  the  marked  nephritic  symptoms,  coupled  with  a 

3«Bang:     Der  Blutzucker,  Wiesbaden,  1913,  p.   128. 

"Neubauer:     Biochem.  Ztschr.,  1910,  vol.  xxv,  p.  284. 

3sRolly  and  Oppermann:     Biochem.  Ztschr.,  1913,  vol.  xlviii,  p.  268. 

3»Hopkins:     Am.  Jour.  Med.  Sc.,  1915,  vol.  cxlix,  p.  254. 

«°Myers  and  Bailey:     Jour.  Biol.  Chem.,  1916,  vol.  xxiv,  No.  2,  p.  147. 


BLOOD    CHEMISTRY   AND   NEPHRITIS  223 

high  creatinine  value  of  4.7,  indicating  that  the  nephritis  had  as 
much  to  do  with  the  cause  of  death  as  the  diabetes.  In  the  three 
fatal  cases  of  diabetes  which  they  studied,  the  first  two  showed  a 
normal  creatinine  value,  with  an  obscure  cause  of  death  in  both, 
scarcely  acidosis  in  their  opinion.  Myers  and  Bailey  reported  in 
this  paper'  a  number  of  cases  of  nephritis  with  as  high  a  blood 
sugar  content  as  0.20  per  cent.  In  four  cases  of  interstitial  neph- 
ritis glycosuria  was  absent,  while  mild  glycosuria  was  present  in 
the  two  cases  of  parenchymatous  nephritis  with  edema.  Many  of 
their  cases  gave  evidence  of  nephritis  complicating  diabetes. 
Mosenthal41  has  recently  emphasized  the  fact  that  cases  of  inter- 
stitial nephritis  secrete  a  urine  of  a  very  constant  low  specific 
gravity  with  low  content  of  chloride  and  nitrogen.  It  is  possible 
that  this  same  factor  may  have  some  influence  on  the  concentra- 
tion of  urinary  sugar.  Myers  and  Bailey  report  one  case  of  1.10 
per  cent  of  blood  sugar,  possibly  the  highest  figure  on  record, 
and  only  0.5  per  cent  in  the  urine.  They  state  that  if  the  neph- 
ritis is  of  the  interstitial  type,  the  data  obtained  for  uncompli- 
cated nephritis  explain  the  elevation  of  the  threshold  point  of 
sugar  excretion  in  these  advanced  cases  of  diabetes.  The  neph- 
ritis may  further  explain  the  difficulty  in  reducing  the  blood  sugar 
of  these  cases  to  normal  by  restrictions  in  the  carbohydrate  in- 
take. The  use  of  lactose  as  a  functional  kidney  test  has  shown 
quickly  the  permeability  of  the  kidney  for  this  sugar  in  nephritis. 
As  an  index  of  the  ability  of  the  kidney  to  excrete  sugar,  it  seems 
possible  that  the  ratio  between  the  sugar  of  the  blood  and  urine 
might  be  worked  out  somewhat  after  the  method  of  McLean,*2 
as  recently  employed  for  urea  and  chlorides. 

Blood  Chemistry  and  Surgery. 

Operative  risk  is  largely  judged  by  kidney  function.  Operative 
risk  means  ability  to  stand  the  anesthetic  and  to  carry  on  the 
functions  in  the  presence  of  an  overwhelming  change  in  the  organ- 
ism caused  by  the  operative  attack.  The  methods  usually  in  vogue 
in  surgical  institutions  to  judge  kidney  function  are  the  routine 
urinary  analysis  and  the  use  of  the  phenolsulphonphthalein  test 
for  kidney  efficiency.  From  what  has  gone  before,  it  seems  ra- 

41Mosenthal :     Arch.  Int.  Med.,  1915,  vol.  xvi,  p.   733. 
^McLean:     Jour.  Exper.  Med.,  1915,  vol.  xxii,  pp.  212,  366. 


224  BLOOD   AND   URINE    CHEMISTRY 

tional  to  include  in  this  survey  of  the  patient  a  very  complete 
blood  chemical  analysis.  Since  the  data  already  obtained  by 
blood  chemical  methods  have  so  often  upset  and  changed  medical 
diagnoses  and  prognoses,  it  goes  without  saying  that  the  same  set 
of  conditions  will  occur  when  these  tests  are  used  in  connection 
with  surgical  procedures.  Certainly  the  surgeon  who  proceeds 
to  operate  after  having  been  assured  that  the  blood  sugar,  urea 
nitrogen,  uric  acid,  and  creatinine  of  his  patient  are  within  nor- 
mal bounds,  will  have  far  less  cause  for  fear  of  unforeseen  catas- 
trophe to  his  patients  than  those  who  rely  simply  on  the  tests  com- 
monly used  with  respect  to  the  urine.  Possibly  in  no  department 
of  surgery  are  these  tests  so  much  indicated  as  in  urology  in  con- 
nection with  operative  procedures  upon  the  old  men-candidates 
for  prostatectomy.  Remarkable  lowering  of  the  death  rate  from 
this  operation  has  occurred  since  the  institution  of  rational  prepa- 
ration of  these  bad  risks  for  surgery  have  been  carried  out,  with 
free  washing  of  the  kidney  for  days  prior  to  the  operation  by 
copious  drinking  of  water,  the  use  of  diuretics,  the  awaiting  un- 
til cardiac  and  renal  functions  are  within  rational  limits  of  health. 
These  patients  are  examined  by  the  routine  methods  of  urine 
analysis,  special  attention  being  paid  to  the  output  of  urea  with- 
out much  attention  to  the  blood  findings.  Estimation  of  urea 
without  blood  urea  determinations  are  necessarily  of  but  little 
scientific  benefit.  These  tests  should  be  supplemented  by  urea 
blood  estimations  as  well  as  blood  sugar  and  uric  acid  and  creatin- 
ine tests. 

Aside  from  the  preliminary  survey  of  these  operative  patients, 
the  surgeon  may  well  utilize  the  methods  of  blood  chemistry  for 
determination  of  the  impending  onset  of  acidosis  in  his  patients 
after  operation.  We  hear  much  of  the  term  acidosis  in  the  surgical 
hospital,  but  hear  but  little  of  its  exact  diagnosis.  Certain  it  is, 
much  that  is  called  acidosis  in  the  way  of  a  surgical  operation  is 
not  acidosis  at  all  and  perhaps  cases  of  acidosis  occur  that  are 
never  recognized.  It  is  here  that  blood  chemistry  must  come  for- 
ward to  settle  this  question.  A  rapid  estimation  of  the  combining 
power  of  the  patient's  blood  plasma  by  the  Van  Slyke  or  Marriott 
method  will  speedily  clear  the  picture  so  far  as  aeidosis  is  con- 
cerned. 


BLOOD   CHEMISTRY  AND   NEPHRITIS  225 

A  recent  study  on  intestinal  obstruction  in  relation  to  the  non- 
coagulable  nitrogen  of  the  blood  is  quite  interesting  along  the  lines 
just  noted.  Cooke,  Rodenbaugh,  and  Whipple'*3  take  up  the  ques- 
tion of  the  analytical  consideration  of  blood  in  cases  of  intesti- 
nal obstruction,  intestinal  closed  loops,  and  other  acute  intoxica- 
tions. Their  interest  in  this  question  was  aroused  by  a  communica- 
tion of  Tileston  and  Comfort,**  who,  in  a  large  series  of  human 
cases,  reported  three  cases  of  intestinal  obstruction  with  very  high 
noncoagulable  nitrogen.  The  present  writers,  Cooke,  Rodenbaugh, 
and  Whipple,  found  that  most  cases  of  intestinal  obstruction, 
especially  with  signs  of  acute  intoxication,  showed  a  high  non- 
coagulable blood  nitrogen,  and  it  seemed  possible  to  them  that  this 
factor  might  be  of  value  in  diagnosis  and  especially  prognosis  of 
acute  abdominal  conditions.  They  have  become  convinced  as  a 
result  of  their  work  that  this  determination  of  nitrogen  in  blood 
is  of  value  in  various  acute  intoxications.  If  the  reading  is  high, 
it  may  be  assumed  that  there  exists  a  dangerous  grade  of  intoxica- 
tion, but  on  the  contrary,  one  may  not  assume  that  a  low  reading 
gives  evidence  of  slight  intoxication,  because  a  fatal  outcome  may 
be  associated  with  a  low  reading.  It  is  therefore  of  considerable 
value  to  know  that  the  noncoagulable  nitrogen  of  the  blood  may 
show  high  readings  in  other  conditions  than  renal  disease.  On 
the  other  hand,  determinations  of  the  blood  urea  alone  are  of 
somewhat  less  value  in  studying  the  retention  products  in  the 
blood  in  these  conditions. 

In  these  animal  experiments  Cooke,  Rodenbaugh,  and  Whipple 
found  that  the  blood  urea  varied  less  than  30  per  cent  to  more 
than  80  per  cent  of  the  total  noncoagulable  nitrogen,  and  while  a 
high  urea  reading  was  the  rule,  the  variations  in  the  urea  curve 
and  the  curves  of  the  other  noncoagulable  nitrogenous  substances 
were  so  great  that  the  urea  reading  was  a  somewhat  unreliable  in- 
dex of  the  extent  to  which  noncoagulable  nitrogenous  substances 
were  retained.  In  these  experiments  dogs  were  used  mainly,  a  few 
cats  and  one  human  case  being  recorded.  The  blood  was  taken  from 
the  jugular  vein  in  some  cases,  from  the  carotid  in  others.  The 
dogs  were  anesthetized  and  loops  of  the  intestine  tied  off,  the 

43Cooke,  Rodenbaugh  and  Whipple:  Jour.  Exper.  Med.,  June,  1916,  vol.  xxiii,  No.  6, 
p.  717. 

"Tileston  and  Comfort:     Arch.   Int.   Med.,   1914,  vol.  xiv,   p.   620. 


226  BLOOD   AND   URINE    CHEMISTRY 

animals  watched,  blood  samples  taken  at  various  intervals;  in 
some  cases  the  dogs  were  reoperated,  in  other  cases  they  were  al- 
lowed to  die  of  their  intoxications  due  to  obstruction  operations. 
Besides  the  animal  experimental  observations,  they  record  one  hu- 
man case  of  intestinal  obstruction,  with  blood  findings. 

These  experiments  showed  definite  increase  in  the  noncoagulable 
nitrogen  in  the  blood  of  cases  of  intestinal  obstruction  with  closed 
loops  of  intestine.  With  acute  intoxication,  the  rise  is  shown  as 
striking  and  constant.  This  rise  was  high  and  was  considered  a 
grave  sign  and  was  a  clinical  index  of  a  severe  intoxication  even 
in  spite  of  the  clinical  evidence  to  the  contrary.  But  a  low 
noncoagulable  nitrogen  does  not  guarantee  a  mild  grade  of  in- 
toxication. Acute  proteose  intoxication  in  animals  due  to  the  in- 
jection of  a  pure  proteose  will  show  a  prompt  rise  in  blood  non- 
coagulable nitrogen,  eA^en  an  increase  of  100  per  cent  within  three 
or  four  hours.  These  intoxications  also  showed  a  high  creatinine 
and  urea  concentration.  The  residual  or  undetermined  nitrogen 
was  also  high.  The  human  case  with  autopsy  showed  the  same 
conditions  as  the  animals  under  experiment.  Clinically  the  non- 
coagulable nitrogen  of  the  blood  may  give  information  of  value 
in  intestinal  obstruction.  A  high  reading  indicates  a  grave  con- 
dition, but  a  low  one  may  still  fail  to  show  a  grave  intoxication. 
The  kidneys  in  all  these  cases  at  autopsy  appeared  normal.  It  is 
possible  that  protein  or  tissue  destruction  rather  than  impaired 
eliminative  function  was  responsible  for  the  rise  in  noncoagulable 
nitrogen  of  the  blood  in  these  acute  intoxications.  Transfusions 
of  dextrose  solutions  often  benefit  intestinal  obstruction  and  may 
depress  the  level  of  the  noncoagulable  nitrogen  in  the  blood.  These 
observers  likewise  state  that  some  cases  show  no  change  in  the 
noncoagulable  nitrogen  following  transfusions  and  diuresis,  and, 
as  a  rule,  such  cases  presented  the  most  severe  intoxication. 

Thus,  another  line  of  investigation  was  opened  up  by  this  blood 
chemical  study  on  intestinal  obstruction.  Perhaps  by  this  kind 
of  research,  the  presence  of  a  severe  and  dangerous  grade  of  sur- 
gical complication  may  be  detected  before  acute  clinical  symptoms 
assert  themselves. 


GENERAL  INDEX 


Accessory  solution  for  test  for  phos- 
phates, in  general  analysis  of 
urine,  107 
Acetone,  test  for,  in  general  analysis 

of  urine,  107 

Acid  sodium  urate  crystals,  122 
Acidosis : 

apparatus  used  in  tests,  61,  64 

bicarbonate  of  sodium  in,  170,  179 

bi chemistry  of,   (Henderson)   192 

consumption  of  fats  injurious  in, 
169 

controls  of  Marriott,  Levy,  and 
Eowntree's  method  for  the 
determination  of  the  hydro- 
gen-ion concentration  of  the 
blood,  69 

definition  of  acidosis  given  by 
Naunyn,  182 

determination  of  the  alkali  reserve 
of  the  blood  plasma,  70 

example  of  reading  on  the  Van 
Slyke  apparatus,  66 

fasting  and  diet  in,  184 

Fridericia's  method  for  determi- 
nation of  carbon  dioxide  in 
alveolar  air,  174 

Henderson  and  Palmer's  experi- 
ments showing  magnitude  of 
alkali,  181 

introduction  of  alkalies  in,  167 

Levy,  Marriott,  and  Bovvntree's 
method  for  the  determina- 
tion of  the  hydrogen-ion  con- 
centration of  the  blood,  66 

lipemia,  181 

nephritis  in,  185 

over  5.0  mgms.  of  creatinine  in 
blood  denotes  fatal  end  of 
any  case,  212 

preparation  of  sacks  for  method,  67 

preparation  of  salt  solution,  71 


Acidosis — Cont  'd 

preparation  of  standard   colors,  67 
producing  acidosis  in  dogs,  183 
results  obtained  in  normal  individ- 
uals,  73 

results  of  study  in  normal  and  path- 
ological  cases,   172 
salt    solution    used    in    method    of 

test,  68 

technic  of  method  for  test,  68 
tests  for,  59 

Van   Slyke   method   for  the    deter- 
mination  of   the   carbon   di- 
oxide    combining    power    of 
the  blood  plasma,  59,  177 
Van  Slyke  method  simplest,  178 
Albumin,    general    analysis    of   urine, 

102 

Heller's  nitric  acid  test,  102 
Robert's  test  for,  103 
Alkali   reserve   of   the  blood   plasma, 

determination  of,   70 
Alumina  cream,  preparation  of,  37 
Ambard  coefficient,  215 
Ammonia : 

aeration  of,  82 
chemicals  used  in,  24 
Ammoniacal-silver-magnesium  mixture 
for  uric  acid  tests,  prepara- 
tion of,  38 

Ammonium  magnesium  phosphate  for 
microscopic  analysis  of  urin- 
ary sediment,  117 

Ammonium  sulphate  solution  for  total 
nitrogen  in  chemical  analysis 
of  urine,  77 
Ammonium    thiocyanate,    preparation 

of,  58 

Ammonium  urate  crystals,  122 
Analysis,  blood  chemical,  28 
Analysis,  general,  of  urine,  96 
Apparatus,    CO2,    showing    air    being 
forced  out  in  tests  for  acido- 
sis of  blood,  64 


228 


GENERAL  INDEX 


Apparatus,  Fridericia,  for  determina- 
tion of  carbon  dioxide  in 
alveolar  air,  173 

Apparatus  for  removing  fumes  in 
..  connection  with  nitrogen  de- 
termination, 48 

Aqueous  solution  of  Napthol  Green 
B  as  a  standard  of  color  in 
cholesterol,  52 


B 


Bacteria,  formula  for  staining,  130 
Beekman  apparatus  for  carrying  out 

cryoscopy,  94          , 
Benedict's    qualitative    solution    for 

glucose  test,  100 
Benedict's   quantative    estimation    of 

glucose,  100 
Benedict's     volumetric    solution     for 

glucose,  101 
Bicarbonate    of    sodium    in    acidosis, 

170,  179 

Bile,  in  general  analysis  of  urine,  107 
Gmelin's  test  for,  108 
Smith's  test  for,  108 
Blood: 

acidosis  in,  61 

casts    in    microscopic    analysis    of 

urinary  sediment,  111,  113 
changes  in  gout,  194 
chemical   analysis    compared    with 

urinary  analyses,  18 
chemistry  and  nephritis,  201 
chemistry,  general  consideration  of, 

17 
estimation  of  sugar  and  creatinine, 

29 

in  general  analysis  of  urine,  108 
benzine  test,  108 
guaiac  test,  108 
manner  of  procuring  and  handling 

25,  26 

pictures  in  gout  and  early  intersti- 
tial nephritis,  199 
pictures  in  gout,  diabetes,  and  ne- 
phritis, 200 

plasma,  saturating  with  carbon  di- 
oxide, 60 
sugar,  139 


Blood — Cont  'd 
sugar  in,  28 

sugar  and  nephritis,  222 
sugar    and    surgery,    necessity    of 
blood  chemical  analysis,  223 
withdrawal  of,  25,  26 
amount  needed,  27 
Gradwohl  method,  27 
Bottles    for   use   in   connection   with 
COs    determination    in    tests 
for  acidosis  of  blood,  62 


Calcium  carbonate  in  microscopic 
analysis  of  urinary  sediment, 
119,  120 

Calcium  oxalate  calculi,  125 
Calcium  oxalate  in  microscopic  analy- 
sis of  urinary  sediment,  118 
Carbon  dioxide,  extracting  in  tests  for 

acidosis  of  blood,  63 
Casts  in  microscopic  analysis  of  uri- 
nary sediment,  110 
Centrifuge,  placing  in  laboratory,  20 
Centrifuge  tube,  50  c.c.,  28 
Centrifuge  tube  attached  to  suction, 

38 

Characteristic  blood  pictures  in  gout, 
diabetes  and  nephritis,  202, 
203 
Chemicals    used   in   blood    and   urine 

chemistry,  22 

Chemical  balance,  placing  of,  in  labo- 
ratory, 20,  25 
Chemicals  blood  bottle,  27 
Cholesterol,  apparatus  used  in  tests, 

24 

chemicals  used  in  tests,  24 
crystals  of,   123 
determination  of,  50 
estimation  of,  with  Hellige  colori- 
meter, 50 

preparation  of  sample,  50 
Chlorides,  57 
example,  58 
example  of  test,  95 
in  chemical  analysis  of  urine,  test 
for,  95 


GENERAL  INDEX 


229 


CO2  apparatus  for  testing  acidosis  in 

blood,  61 
CO2    apparatus    showing    air    being 

forced  out  in  aeidosis  test,  64 
Color  of  urine  in  general  analysis  of 

urine,  96 

Colorimeter,  description  of,  132 
Congo  red  used  in  determination  of 

total  nitrogen,  55 
Creatine     and     creatinine,     chemicals 

used  in  solution  of,  23 
Creatinine : 

estimation    in    blood    with    Hellige 

colorimeter,  34 
in  chemical  analysis  of  urine,  test 

for,  88 

standard  solution  of,  35 
Cryoscopy  of  blood  and  urine,  92 
Cylindroids  in  microscopic  analysis  of 

urinary  sediment,  114 


Definition  of  acidosis  given  by 
Naunyn,  182 

Description  of  colorimeter,  132 

Determination  of  alkali  reserve  of 
blood  plasma,  70 

Determination  for  total  nitrogen,   76 

Determination  for  total  solids,  53 

Development  of  color  in  tests  in  chem- 
ical analysis  "of  urine,  80 

Development  of  color  in  urea  tests, 
44 

Diabetes  phlorizin,  145 

Diacetic  acid  in  general  analysis  of 

urine,  105 
Gerhardt's  test,  105 

Diagram  illustrating  excessive  sugar 
formation  through  retention 
of  glycogen  in  liver,  144 

Diagram  illustrating  normal  sugar 
embolism,  140 

Diagram  showing  assimilation  of  sug- 
ar in  diabetes,  143 

E 

Epithelial  tests  in  microscopic  analy- 
sis of  urinary  sediments,  111, 
112 


Erythrocytes   in  microscopic  analysis 

of  sediment  in  urine,  115 
Estimation  of  blood  sugar  with  Hel- 
lige colorimeter,  table  I,  31 

cholesterol,   table   V,   51 

creatinine  in  blood,  table  II,  34 

creatinine  in   chemical   analysis  of 
urine,  table  VII,  64 

nitrogen,  table  IV,  45 

phenolsulphonphthalein,  table  VIII, 
91 

protein,    in     general     analysis     of 
urine,  103 

total  nitrogen,  table  VT,  78 

uric  acid,  table  III,  40 
Estimation     of     freezing     point     of 

blood,   92 

Examining  urinary  sediment  for  sim- 
ple organisms,  129 

Example   of,    estimation   of    nitrogen 
with  Hellige  colorimeter,  45 

Mohr  method  of  determining  chlo- 
rides, 58 

reading  in  cholesterol,  51 

reading  on  Van  Slyke  's  apparatus 
for  acidosis  of  blood,  66 

readings  of  cryoscopy,  93 

result  in  using  standard  solution  of 
creatinine,  35 

sugar  in  blood,  31 

test  for  creatinine  in  chemical  anal- 
ysis   of    urine,    88 

test  for  glucose,  102 

test  for  phosphates,  107 

test  for  specific  gravity  of  normal 
urine,  98 


Fasting  and  diet  in  acidosis,  181 
Fatty  casts  in  microscopic  analysis  of 

urinary  sediments,  113 
Fifty  c.c.  centrifuge  tube,  28 
Finding  over  5.0  mgms.  of  creatinine 

in  blood  denotes  fatal  end  in 

acidosis,  212 
Folin-Farmer    microchemical    method 

in  total  nitrogen,  56 
Folin-Macallum  reagent  in  uric  acid 

tests,  39,  84 


230 


GENERAL  INDEX 


Foreign  substances  due  to  contamina- 
tion, microscopic  analysis  of 
urinary  sediment,  116 

Formula  for  preparation  of  sodium 
carbonate,  29 

Fridericia  apparatus  for  determina- 
tion of  carbon  dioxide  in  al- 
veolar air,  173 


G 


Gentian  violet  stain  for  staining  bac- 
teria, formula  for,  130 
Gerhardt's  test  for  diacetic  acid,  105 
Glucose,  in  general  analysis  of  urine, 

100 

qualitative  test  for,  100 
qualitative  solution  for,  100 
quantitative  estimation  of,  100 
volumetric  solution  for,  100 
Janney's     studies     of     formations 

from  body  protein,  146 
Gmelin's  test  for  bile,   108 
Gout,  advisability  of  blood   chemical 
analysis  in  dealing  with  sus- 
pected cases,  200 
amount  of  uric  acid  under  normal 

conditions,  194 

differentiating   gout   from   rheuma- 
tism and  other  joint  affairs, 
197 
increase  in  uric  acid  concentration, 

196 
repeated     examinations     necessary, 

196 

uric   acid   can   be   found  in  blood 
without  gouty  symptoms,  198 
Graduated  centrifuge  tube  used  in  de- 
termining chlorides,  57 
Graduated   sugar   tube,  29 
Gradwohl's  tourniquet,  26,  27 
Granular  casts  in  microscopic  analysis 
of    urinary    sediments,    110, 
111 

Guaiac  test  for  blood  in  general  anal- 
ysis of  urine,  108 

H 

Hellige  colorimeter: 

choice  for  practical  work,  the,  132 


Hellige  colorimeter — Cont  'd 
description  of,  132 
estimation    of    blood    sugar    with, 

table  I,  31 

,    cholesterol  with,  table  V,  51 
creatinine  in  blood  with,  table  II, 

34 
creatinine  in  chemical  analysis  of 

urine,  table  VI,  86 
nitrogen,  table   IV,  45 
phenolsulphonphthalein,   table 

VIII,   91 
protein,    in    general    analysis    of 

urine,  table  X,  104 
total  nitrogen,  table  VI,  78 
uric   acid,   table   III,   40 
optical  arrangement  of  window  in, 

137 
representations  of,  133,  134,  135, 

136 

Henderson  and  Palmer's  experiments 
showing  magnitude  of  alkali, 
181 

Hippuric  acid  crystals,  124 
Hyaline  casts  in  microscopic  analysis 
of    urinary    sediments,    111, 
112 

Hydrogen-ion  concentration  of  the 
blood,  Marriott,  Levy,  and 
Eowntree's  method  of  deter- 
mining, 66 


Indican,  Obermasyer's  test  for,  106 

Indicator,  ferric  alum  in  determina- 
tion of  chlorides,  57 

Indigo-carmin  test  for  kidney  effi- 
ciency, 91 

Interpretation  of  results  from  Mar- 
riott, Levy,  and  Eowntree's 
experiments  in  acidosis  of 
blood,  74 

Installation  of  blood  and  urine  labo- 
ratory, 19 

Introduction  of  alkalies,  method  of, 
167 


Janney  technic  in  cases  of  phlorizin 
diabetes,  145 


GENERAL   INDEX 


231 


Kidney  efficiency,  indigo-carmin  test 
for,  91 

Kidney  function  in  operative  risk, 
223 

Kidney,    tuberculous,    treatment.    128 

Kjeldahl  apparatus  showing  conden- 
ser in  total  nitrogen,  55 

Kjeldahl  flask  for  determination  of 
total  nitrogen,  54 


Laboratory,  blood  and  chemical.  19 
installation  of,  19 
selection  of   room,   20 
views  of,  21,  22,  23 

Leucine  crystals,   124 


Marriott,  Haessler  and  Rowland's 
method  in  estimating  aeido- 
sis  in  nephritis,  185 
Marriott  and  Rowland's  method  of 
estimating  acidosis  in  ne- 
phritis, 185 

Marriott,      Levy,      and      Eowntree's 
method  of   the  hydrogen-ion 
concentration  of  the  blood: 
apparatus  required,  71 
comparison    of    tubes    with    stand- 
ards, 69 

controls  of  method,  69 
preparation  of  sacks,  67 
preparation  of  standard  colors  (ac- 
cording to  Sorenson),  67 
salt  solution  used  in  method,  68 
technic  of  method,  68 
Method    of    determination    in    alkali 
reserve  of  the  blood  plasma, 
72 
Method    of    introduction    of    alkalies 

in   acidosis,  167 

Method  of  washing  sacks  used  in  aci- 
dosis tests,   72 
Microburner,  47 

Microscopic  analysis  of  urinary  sedi- 
ment: 

centrifuge  for,  109 
ronical  centrifuge  tube  for,  109 


Microscopic  analysis  of  urinary  sedi- 
ment— Cont  'd 
organized   sediments,   110 
casts,  110 
blood,  111 
epithelial,  111 
fatty,  113 
granular,  110 
hyaline,  111 
pus,  114 
waxy,  113. 
cylindroids,   114 
erythrocytes,  115 
fibrin,  116 

foreign    substances    due    to    con- 
tamination, 116 
spermatazoa,  116 
tissue  debris,  116 
urethral  fragments,  116 
pathological  condition  in  which  leu- 
cine  and  tyrosine  have  been 
found,   124 
pathological      conditions    in    which 

uric  acid  is  found,  121 
preparation  of  sediment,  109 
unorganized  sediments,  116 

ammonium  •magnesium  phosphate, 

117 

calcium  carbonate,    119 
calcium  oxalate,  118 
calcium  phosphate,  118 
calcium  sulphate,    119 
cholesterol,   123 
cystine,  123 
hippuric  acid,  124 
leucine  and  tyrosine.  124 
urates,  123 
uric  acid,  121 
urinary  calculi,   125 
Modification    of    test    for    nonprotein 
nitrogen  to   serve   for  blood 
estimations,  47 

Mohr    method    in    determining    chlo- 
rides, 58 


X 


Napthol    Green   B   as    a   standard  of 
color  in  cholesterol,  52 


232 


GENERAL  INDEX 


Nephritis: 

Ambard's  coefficient,  215 
blood  chemical  figures  most  trust- 
worthy, 209 
blood  sugar  in,  222 
blood  picture  of  gout,  diabetes,  and 

nephritis,  202,  203 
cases  of  thermic  fever,  reports  of, 

212 
death  rate  lower  in  surgery  after 

'  treatment  for  kidneys,  224 
importance  of  creatinine  in  routine 
blood    chemical    analysis    in 
connection  with  chronic  ne- 
phritis, 205 
McLean's  modification  of  Ambard's 

coefficient,   221 

phenolsulphonphthalein  in,   208 
scale  of  degree  of  impairment  of 
renal   function   as   indicated 
by  the  tests  employed,  219 
table  of  blood  and  urine  findings  in 

thermic   fever,   213 
table   of   uric   acid,   nitrogen,   and 
creatinine  of  blood  in  inter- 
stitial  nephritis,   211 
j    test  meal  for   renal   function   and 

Ambard's  coefficient,  215 
total  nitrogen,  201 
valuable  report  of  unusual  case  of 
chronic  interstitial  nephritis, 
214 

value  of  Ambard's  coefficient,  220 
value    of    Geraghty    and    Rowntree 

test,  207 

Nessler's  solution,  preparation  of,  44 
Nessler's  solution  for  total  nitrogen, 

47 
Nitric    acid    ring   test    for    albumin, 

102 
Nitrogen,  estimation  of,  with  Hellige 

colorimeter,    78 
Nonprotein  nitrogen,   chemicals   used 

in,  24 

Nonprotein,   modification   of   test    to 

serve  for  blood  estimates,  47 

Normal  urine,  appearance  of,  96 

odor  of,  97 

reaction,  98 


Normal  urine — Cont  'd 
specific  gravity,  98 


Obermayer's  test  for  indican,  106 

Odor  of  normal  urine,  97 

Optical    arrangement    of    window    of 

colorimeter,  137 
Organized    sediments    in    microscopic 

analysis  of  urinary  sediment, 

' 


Pathological  conditions  in  which  ex- 
cretion of  potash  is  in- 
creased, 106 

Pathological  conditions  in  which  leu- 
cine  and  tyrosine  have  been 
found,  121 
Pathological  conditions  in  which  uric 

acid  is  found,  121 
Phenolsulphonphthalein : 
apparatus  used  in,  24 
chemicals  used  in,  24 
estimation  of,  91 
example  of  test,  91 
graduated  syringe  used  for  injec- 
tion of,  90 

preparation  of  solution,  89 
procedure,  89 
standard   preparation,   90 
use  of,  in  nephritis,  208 
Phlorizin    diabetes,    Janney's    experi- 
ments,  146 
Phosphates : 

accessory  solution  for,  107 
example  of  test  for,  107 
pathological  condition  in  which  the 

excretion  is  decreased,  106 
pathological  condition  in  which  the 

excretion  is  increased,  106 
Picramic  acid  solution,  standard,  30 
Preparation    of    Folin-Macallum    re- 
agent in  uric  acid  test,  39 
Preparation    for    indicator    used    in 
chlorides    in    chemical    anal- 
ysis of  urine,  95 


GENERAL  INDEX 


233 


Preparation  of  phosphate  mixture  in 
determination  of  alkali  re- 
serve of  blood  plasma,  71 

Preparation  of  sacks  for  tes-t  in  acid- 
osis  of  blood,  67 

Preparation  of  salt  solution  for  de- 
termination of  alkali  reserve 
of  blood  plasma,  71 

Preparation  of  sodium  carbonate, 
formula  for,  29 

Preparation  of  sodium  hydroxide  in 
total  nitrogen  determina- 
tions, 56 

Preparation  of  sodium  standard  colors 
for  comparison  of  color  in 
carrying  out,  tests  for  acid- 
osis  in  blood,  67 

tests  for   acidosis  with  pure   chol- 
esterol, 51 

tests  for  acidosis  uric  acid,  stand- 
ard solution,  84 

Producing  acidosis  in  dogs  for  exper- 
imental purposes,  183 

Protein,  Janney's  studies  from  glu- 
cose formation  from  body 
protein,  144 

quantitative     estimation     (Purdy), 
103 


Qualitative    test    for    glucose,    Bene-  j 

diet's,  100 
Quantitative    estimation    of    glucose,  j 

Benedict's,   100 
Quantitative     estimation    of     protein  j 

(Purdy),  103 


R 

Reaction  of  normal  urine,  98 

Renal  diabetes  in  pregnancy,  151 

Renal  test  meal,  215 

Representation  of  Hellige  colorim- 
eter, 133 

Robert's  test  for  albumin,  103 

Robert's  reagent  for  nitric  acid  test 
for  albumin,  103 

Roux's  blue,  formula  for,  130 


Salt  solution  used  in  method  of  Mar- 
riott, Levy,  and  Rowntree  for 
acidosis  in  blood,  68 
Saturating  blood  plasma  with  carbon 

dioxide,  60 
Sediments  in  microscopic  analysis  of 

urine 
examining    for    simple    organisms, 

129 

organized  sediments,  110 
unorganized  sediments,   116 
Smith's  test  for  bile,  107 
Sodium  hydroxide,  preparation  of,  56 
Solids  of  normal  urine,  98 
Solution   of    ammonium   sulphate,   42 
Solution,  Nessler's,  44 
Specific  gravity  of  normal  urine,  98 
Spermatazoa  in  microscopic   analysis 

of  urinary  sediment,  116 
Staining  of  bacteria  in  urine,  128 
bacillus  tuberculosis,  128 
bacillus  typhosus,  128 
carbol  gentian  violet  for  modifica- 
tion  of,    Gram  method,   130 
diagnosis  of  tuberculosis  from  urin- 
ary sediment  important,  128 
Roux's  blue  for  simple  organisms, 

129 

test  for  bacillus  tuberculosis,  128 
Standard  solutions: 

ammonium  thiocyanate,  95 
bichromate  of  potash,  55 
phenolsulphonphthalein,  90 
silver  nitrate,  95 
sodium  carbonate,  29 
uric  acid,  84 
Sugar  in  blood: 

advisability  of  beginning  blood 
chemical  analysis  at  once 
with  sugar  and  creatinine  be- 
cause of  their  quick  dete- 
rioration, 26 

best  time  to  test  for,  160 
cases  in  literature,  153 
diabetes  mellitus,  159 
estimations  of,  with  Hellige  color- 
imeter, 31 
example  of  readings,  31 


234 


GENERAL  INDEX 


Sugar  in   blood — Cont'd 
graduated  sugar  tube,  29 
Gradwohl  data  on  blood  and  urine 

cases,  160 

Ostwald  pipette  in,  29 
picramic  acid  solution  in,  30 
renal  diabetes  in  pregnancy,  151 
saturated   solution   of   sodium  car- 
bonate, 29 
sugar  tube  immersed  in  beaker  of 

water  in  test,  30 
Surgery,  blood  chemistry  and,  223 


T 


Table  for  estimation  of  blood  sugar 
with  Hellige  colorimeter, 
table  I,  31 

cholesterol,  table  V,  51 
creatinine  in  blood,  table  II,  34 
creatinine   in   chemical   analysis   of 

urine,  table  VI,  86 
nitrogen,  table  IV,  54 
phenolsulphonphthalein,    table 

VIII,  91 
protein,     in    general     analysis     of 

urine,  table  X,  104 
total  nitrogen,  table  VI,  78 
uric  acid,  table  III,  40 
Table  showing,  blood  pictures  of  gout 
and  early  interstitial  nephri- 
tis, 199 

scale  of  degree  of   renal  function 

by  tests  employed  in  blood 

chemistry  and  nephritis,  219 

Technic  of  Marriott,  Levy,  and  Rown- 

tree,  68 

Test  meal  for  renal  functions  in  blood 
chemistry  and  nephritis,  215 
Test  with  guinea  pigs  for  renal  tuber- 
culosis, 129 
Tests  for  bile  in  general  analysis  of 

urine,  108 
Tissue  debris  in  microscopic  analysis 

of  urinary  sediments,  116 
Toluene    satisfactory    for    preserving 

urine  for  test  purposes,  99 
Total  nitrogen: 
determination,  76 


Total  nitrogen — Cont'd 

digestion  rack,  55 

estimation  of,  with  Hellige 's  color- 
imeter,   78 

example,  78 

Folin-Farmer    microehemical   meth- 
od, 56 

Kjeldahl  apparatus,  55 

Kjeldahl  flask,  54 
Total  solids: 

calculation  in,  53 

determination,  53 

weighing  bottle  for,  53 
Tourniquet,     Gradwohl 's,     for     blood 

withdrawal,  26,  27 
Tube,  50  c.c.  centrifuge 


U 


Unorganized  sediments  in  microscopic 
analysis     of     urinary     sedi- 
ments, 116 
Urates,  123 

Urea,  apparatus,  arrangement  for,  22 
apparatus,     set    up   and    connected 

with  suction,  43 
development  of  color,  44 
estimation  of  nitrogen  in  with  Hel- 
lige colorimeter,  45 
result,  80 
Urea  N.,  apparatus  used  for,  22 

chemicals  used  in,  22 
Urease,  where  obtainable,  42 
Urethral    filaments,    in    microscopical 
analysis     of     urinary     sedi- 
ments, 116 
Uric  acid: 

apparatus  used  in,  23 
chemicals  used  for,  23 
crystals    of,    121 
solution,   preparation    of,    39 
test  for,  84 

Uric  acid  and  urate  calculi,  125 
Uric  acid,  urea  nitrogen,  and  creatin- 
ine   of    blood    in    interstitial 
nephritis,  211 
Urinary  sediments: 

microscopic  analysis,  109 
organized,  110 


GENERAL   INDEX 


235 


Urinary  sediments — Cont  'd 
preparation  of,   109 
unorganized,  116 
Urinary  calculi,  125 
murexide  test  for,  126 
table  illustrating,  127 
Urinary  analyses  compared  with  blood 

analyses,  18 
Urine : 

color  of  normal,  96 
example   of    determination    of    spe- 
cific gravity,  98 
Long's  coefficient,  98 
pathological  conditions  which  cause 

decrease  of  output  of,  96 
pathological  conditions  which  cause 

increase  of  output  of,  96 
reaction  of  normal,  97 
separate    day    and    night    urine    in 

pathological  cases,  99 
specific  gravity  and  solids,  98 
table  of  color,  cause  of  coloration 

and   pathological    conditions, 

97 

transparency  of,  96 
volume,  96 


Value  of  toluene  for  preserving  urine 
for  testing,  99 


Van  Slyke's  carbon  dioxide  appara- 
tus, arrangement  of,  21 
Van  Slyke  's  method  for  the  determin- 
ation of  the  carbon  dioxide 
combining  power  of  the  blood 
plasma,  59 

apparatus   showing  operator   satur- 
ating blood  plasma  with  car- 
bon dioxide,  60 
CO2  apparatus,  61,  65 
CO2   apparatus    showing    air    being 

forced  out,  64 

extracting  carbon  dioxide,  63 
dropping  bottles  used  in,  62 
Volhard-Arnold  method  of   determin- 
ing  chlorides,  57 
Volume  of  urine  in  general   analysis 

of  urine,  96 

Volumetric   flask   used   in   developing 
color  in  uric  acid  test,  39 


W 


Washing  sack  used  in  tests  for  acid- 

osis,  method  of,  72 
Waxy   casts    in   microscopic    analysis 

of  urinary  sediments,  113 
Weighing  bottle  for  total  solids,  53 


AUTHORS  INDEX 


ABDERHALDEX,  32 

ADDIS  AXD  WATAXABA,  220,  221 

ADLER  AND  RAGLE,  194 

AGXEW,  32,  49 

ALDEHOFF,  142 

ALLEX,  32,  142,  153,  162,  164,  181 

AMBAKD,  155,  217 

ABTHAUD,  142 

Arsnx  AXD  Mn.T.FR,  49 

ArTEXRIETH   AM)  FUXK,  52 

AXD  KOEXIGSBEBGER,  133 


BAXG,  32,  52,  222 

BAOIAXXJ  205 

BEDDARD,  PEMBERT,  AXD  SPEIGGS.  75 

BENEDICT,  100,  141,  155,  159 

BEXEDICT  AXD  HITCHCOCK,  41 

BENEDICT  AXD  LEWIS,  17 

BEBXAKD,  148 

BERTBAXD,  32 

BIEBEY,  32 

BLUMEXTHAL.  158 

BLOCK,  50,  52 

BOCK  AXD  BEXEDICT,  49 

B6K,  32 

BOOTHBT  AXD  PEABODT,  75,  177 

BRUGSCH  AXD  SCHITTEXHEM,  192 

BTTTE,  142 


CAPARELLI,  142 

CHACE  AXD  MYERS,  35,  41,  46,  210, 

211,  220 
CHELLE,  32 
COMBE  AXD  LEYI,  46 
COOSE,  BODEXBATTGH,  AND  WHtPPLE, 

225 

COXTEJEAX,  150 
COOLEX,  150 
CBEMER  AXD  RrrrEB,  150 


DAKTX  AXD  DUDLT,  56 
DE  DOMIXICIS.  142 
DE  LAXGEX,  153 
DE  Rncn,  142 
DOBXEB,  32,  205 
DU  SABLOX,  147 


ECKHAKD,  148 
32 


FAXDERX,  32 

FABB  AXD  Arsnx,  32,  49 

FARE  AXD  EBTUBHABB,  32,  49 

FABB  AXD  WILLIAMS,  49 

FETTT.TXG,  155, 159 

FKHT.TXG  AXD  PTRDT.  100 

FLXE,  197,  199,  200 

FIXE  AXD  CHACE,  41 

Frrz,49 

FLATOW,  32 

Foux,  17,  32,  35,  49,  141,  155.  2 

Foutx  AXD  DEXIS,  17,  32.  35.  41. 

49,  56.   194,  195,  19S,   9 

205,  206 

FOLEK,  DEXIS,  AXD  SETMOUB,  32,  4S 

Foux  AXD  FARMXB,  49,  56,  76 
FOLIX,  KABSXEB,  AXD  DEXIS,  32 
FOLD?  AXD  LTMAX,  49 

FOLD?    AXD    MACALLOt.    41 

Foux  AXD  PETTIBOXE,  46 

FOLKXER  AXD   JOSEPH,   91 

FOSTER,  35,  46,  49.  139,  147,  151,  £ 
FBAXK,  32,52 
FBAXK  AXD  TSAAK,  32 
FKOESICIA,  174 
FROTHIXGHAM.  49,  1S9 
FROTHEXGHAV.     Firr,     Foux,    A 
DEXIS,  32,  207 

FBOTHEKGHAlt  AXD  SlOLLIE,  49 


238 


AUTHOR'S  INDEX 


GAGLIO,  142 

GARDNER  AND  MCLEAN,  32 

GARROD,  194,  197 

GERAGHTY  AND  ROWNTREE,  132,  207 

GERHARDT,  105 

GILBERT,  32 

GLEY,  142 

GMELIN,  108 

GRADWOHL,  25,  26,  149 

GRADWOHL  AND  BLAIVAS,  47,  49,  76 

GRAHAM,  153 

GREENWALD,  49 

GRIESBAU,  32 

GRIGANT,  52 

GULICK,  49,  56 


HAGELBERG,  32 

HALSEY,  214 

HAMMANN  AND  HIRSCHMANN,  139 

HANES,  52 

HARDING  AND  WARENEFORD,  49 

HARLEY,  142 

HAWK,   32,  111,   112,  113,  115,  120, 

122,  124 

HECHT-GRADWOHL,  162 
HEDON,  142 
HELLER,  102 

HENDERSON,  66,  165,  166,  189,  192 
HENDERSON  AND  PALMER,  185 
HENES,  52 

HENSEL  AND  WEIL,  120 
HERTER,  149 
HERTZ,  49 
HIGGINS,  75,  177 
HIGGINS  AND  MEANS,  75 
HIGGINS,  PEABODY,  AND  FITZ,  75 
HOHLWEG,  49 
HOPKINS,  32,  222 
HOPKINS  AND  JAMES,  49 
HORNER,  174 

HOWLAND,  179 

ROWLAND,  HAESSLER,  AND  MARRIOTT, 
185 

HOWLAND   AND   MARRIOTT,    73,    75,    166 


JAKSCH,  114 

JANNEY,  144,  145 

JONES  AND  AUSTIN,  221 

JOSLIN,  32,  141,  148,  151,  159,  179 


KARSNER  AND  DENIS,  32,  49 

KJELDAHL,  155 

KLEEN,  159 

KLEMPERER,  150 

KLERCKER,  204 

KOLISH,  150 

K*ORANYI,  92 

KRISTELLER,  46 

KUELZ,  144 

KUELZ  AND  WRIGHT,  150 

KUEMMEL,  93 
KUMAGAVA  SUTO,   32 
KUTSCHMER,  149 


LANCEREAUX,  142 

LEPINE,  150 

LEVENE,  150 

LEVY  AND  ROWNTREE,  75 

LEWIS  AND  BENEDICT,  32,  155 

LEWIS   AND   MOSENTHAL,    152 

LEWIS,  RYFFEL,  AND  OTHERS,  185 

LlEFMANN  AND   STERN,   32 
LlFSCHUTZ,  52 
LOEFFLER,  130 
LOWY,  49 
LUSK,  145,  150 

M 

MACLEOD,  32 
MAGNUS,  32 
MAGUITZ,  32 
MARFAN,  TOBLER,  AND  HELMHOLZ, 

168 

MARKUSE,  142,  150 
MARRIOTT,  70,  74,  75,  165,  224 
MARRIOTT  AND  HAESSLER,  186 
MARRIOTT  AND  HOWLAND,  185 
MARRIOTT,  LEVY,  AND  ROWNTREE,  66, 

75,  171 


AUTHOR'S  INDEX 


239 


MARSHALL,  46 
MARSHALL  AND  DAVIS,  32 

McCLENDON,    75 

McCLENDON   AND    MAGOON,    75 

MCLEAN,  223 

MCLEAN  AND  SELLING,  32,  49,  217 

McLESTER,    195 
MlCHAELlS   AND   EONA,   32 
MlCHAND,   49 

MINKOWSKI,  142,  150 

MOECKEL  AND    FRANK,   32 

MOHR,  57,  145 

MORITZ  AND  PRAIJSNITZ,  150 

MOSENTHAL,  49,  223 

MOSENTHAL  AND  LEWIS,  216,  218 
MULLER,  32 

MYERS  AND  BAILY,  32,  222 

MYERS  AND  FINE,  31,  32,  34,  35,  40, 
41,  45,  49,  51,  52,  53,  56,  57, 
76,  78,  86,  91,  100,  204,  205, 
206 

MYERS,  FINE,  AND  LOUGH,  197,  198 

MYERS  AND  LOUGH,  35,  210 

N 

NAUNYN,  33,  156 
NEUBAUER,  33,  149,  222 
NEUMANN,  46 


OBERMAYER,  106 
OGDEN,  117,  125 
OLIVIERI,  46 


PALLADIN  AND  WALLENBURGER,  205 

PAVY,  33,  150 

PEABODY,  75 

PEARCE,  33 

PEPPER  AND  AUSTIN,  49,  221 

PEYER,   111,   113,  114,  122 

PLASS,  49. 

PLESCH,  75,  177 

PRATT,  195,  196 

PRIBRAM,  49 

PURDY,  103 


BEACH,  196 

EEALE,  142 

EEICHER  AND  STEIN,  33 

EEMOND,  142 

EIESSER,  205 

EGBERT,  103 

EOLLY  AND  OPPERMANN,  33,  222 

EOSE    AND    COLEMAN,    46 
EOWNTREE,   189,   193 


SANDMEYER,  142 

SCHABAD,    142 
SCHIFF,  149 

SCHIROKAUER,  33 

BCHLUTZ  AND  PETTIBONE,  49 

SCHMIDT,  52 

SCOTT,  33 

SEELIG,  142 

SHAFFER,  33,  155,  205,  206 

SlEBECK,    46 

SMITH,  108 
SORENSON,  66 
STEN STROM,  33 
STILLING,  33 
STILLMAN,  75 
STROUSE,  33 


TACHU,  33 

TAKATASCHI,  33 

TAYLOR  AND  HUTTON,  33,  49,  157,  158 

TAYLOR  AND  LEWIS,  49 

THIROLOIX,  142 

TlLESTON   AND    COMFORT,   225 
TlLESTON  AND   HUTTON,   33 


VAN  SLYKE,  59,  74,  75,  174,  176,  192 

VAN  SLYKE  AND  CULLEN,  46,  221 

VOGT,  196 

VOLHARD- ARNOLD,  57 

VON  HESS,  33 

VON  HOOGENHUYZE    AND    VERPLOEGH, 

204 
VON  JAKSCH,  197 


240 


AUTHOR'S  INDEX 


VON  MERINO,  142,  150 

VON  MERINO  AND  MINKOWSKI,  144 

VON  NOORDEN,  33,  142,  143,  197 

W 

WALKER  AND  FROTHINGHAM,  177 
WALPOLE,  101 
WEILAND,  33 
WEINTRAUB,  142 
WEISS,  41 


WESTON,  52  s 

WESTON  AND  KENT,  52 
WINDAUS,  52 
WOLF  AND  SHAFFER,  204 
WOODS,  35,  49 

WOODYATT,  33,  150,  189,  191 
WOODYATT,    SANSUM,    AND    WILDER, 
158 


ZUNTZ,  150 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 

Los  Angeles 
This  book  is  DUE  on  the  last  date  stamped  below. 


J3iomedical  Library 
'omedical  Library 

in* 1991 

EP  21 


Form  L9-5m-3,'67(H738i,8)4939 


UC  SOUTHERN  REGIONAL  LIBRARY  FACILITY 


A    001  286148    o 


